Cationic steroid antimicrobial diagnostic, detection, screening and imaging methods

ABSTRACT

The invention relates to diagnostic, detection, screening and imaging methods. In various embodiments, methods of diagnosis, detection, screening and imaging include administering a cationic steroid antimicrobial or CSA to a subject having or at risk of having an infection or a hyperproliferative disorder (e.g., a tumor, cancer or neoplasia) in an amount effective to diagnose or detect the infection or the hyperproliferative disorder (e.g., a tumor, cancer or neoplasia) in the subject. In a particular aspect, a detectable CSA, namely CSA-13 labeled with 99mTc is used to detect the presence of an infection.

TECHNICAL FIELD

The invention relates to diagnostic, detection, screening and imagingmethods. In various embodiments, methods of the invention includeadministering a cationic steroid antimicrobial or CSA to a subjecthaving or at risk of having an infection or a hyperproliferativedisorder (e.g., a tumor, cancer or neoplasia) in an amount effective todiagnose or detect, diagnose, screen for or image the infection or thehyperproliferative disorder (e.g., a tumor, cancer or neoplasia) in thesubject.

INTRODUCTION

Ceragenins (cationic steroid antimicrobials or CSAs) are syntheticallyproduced small molecules that display broad spectrum antibacterialactivity. These compounds are derived from a steroid backbone appendedwith amino groups, amino acids and other chemical groups (Savage P. B.,et al., FEMS Microbiology Letters (2002) 217:1). Specific ceragenins arehighly bactericidal and while others effectively permeabilize the outermembranes of Gram-negative bacteria (Li C., et al., Antimicrob AgentsChemother 43:1347 (1999), (Schmidt E. J., et al., J Antimicrob Chemother47:671 (2001)). Ceragenins exhibit various levels of selectivity formembranes of eukaryotes vs. prokaryotes (Ding B., et al., J Med Chem45:663 (2002)). These compounds have a net positive charge that iselectrostatically attracted to the negatively charged cell membranes ofcertain viruses, fungi and bacteria.

Ceragenins have a high binding affinity for such membranes (includinglipid A) and are able to rapidly disrupt the target membranes leading torapid cell death. While ceragenins have a mechanism of action that isalso seen in antimicrobial peptides, which form part of the body'sinnate immune system, they avoid many of the difficulties associatedwith their use as medicines. They are simple to prepare and are notsubstrates for proteases. In vitro studies show that ceragenins areactive against a wide range of viral, fungal, and bacterial targetsincluding those resistant to current therapies. The antibacterialproperties of a ceragenin, CSA-13 (FIG. 1) (Savage P. B., et al., FEMSMicrobiology Letters (2002) 217:1), against vancomycin-resistantStaphylococcus aureus, vancomycin intermediate resistant S. aureusstrains, vancomycin-resistant Enterococci, and methicillin-resistant S.aureus, as well as key

Gram-negative pathogens such as Pseudomonas aeruginosa and Escherichiacoli, and bioterrorism surrogate strains for Anthrax, Listeria andplague have also been reported. CSA-13 also inhibit HIV infection ofprimary human CD4+ T cells, the virus's in vivo targets. The reportsindicate that CSA-13 most likely attacks the viral membrane and disruptsthe virus from interacting with its target cells, similar to some of theknown microbicidal peptides. This is important, as a compound thattargets the viral membrane is likely to be effective against all strainsof the virus, regardless of mutations, as the viral membrane remainsunchanged.

Ceragenin CSA-13 is not to be toxic to epithelial cells, keratinocytesand fibroblasts at concentrations significantly higher than thoserequired for bactericidal and virucidal activity. CSA-13 is indefinitelystable as a solid and in solution form and may be stored at roomtemperature or in refrigerator (Savage P. B., et al., Eur J Org Chem 759(2002)).

SUMMARY

The invention provides methods and kits for diagnosis, detection,screening and imaging, in vitro, ex vivo and in vivo. Methods include,among other things, administering a cationic steroid antimicrobial orCSA (e.g., a detectable or labeled CSA) to a subject having or at riskof having an infection or a hyperproliferative disorder (e.g., a tumor,cancer or neoplasia) in an amount effective to diagnose, detect, screenfor or image the infection or the hyperproliferative disorder (e.g., atumor, cancer or neoplasia) in the subject. A direct method of labelingof ceragenins with ^(99m)Tc is disclosed which is simple, rapid, andefficient. The labeled ceragenin, CSA-13 (FIG. 1), contains multipleamine groups, and it is expected that labeling occurs via associationwith one or more of the amine groups.

DESCRIPTION OF THE DRAWINGS

FIG. 1: Structures of CSA-13, CSA-107 and CSA-110.

FIG. 2: Effect of pH on labeling efficiency of ^(99m)Tc-CSA (n=4 perstudy).

FIG. 3: Effect of reducing agent SnCL.2H₂O amount on the labelingefficiency of ^(99m)Tc-CSA (n=4 per study).

FIG. 4: Rate of complexion of ^(99m)Tc with CSA-13 and stability of^(99m)Tc-CSA (n=4).

FIG. 5: Paper electrophoresis of ^(99m)Tc-CSA and ^(99m)TcO₄ ⁻ in sodiumphosphate buffer of pH: 6.8 using Whatman 1 as support. The samples wererun at constant voltage of 300V for 1 hour.

FIG. 6: In-vitro binding of ^(99m)Tc-CSA to viable S. aureus (n=4 perstudy).

FIG. 7: In-vitro binding of ^(99m)Tc-CSA to viable S. aureus incomparison with ^(99m)Tc-Ciproflaxin at 50 μg concentration (n=4 perstudy).

FIG. 8: Scintigram of a rat at 4 hours after administration of^(99m)Tc-CSA showing S. aureus lesion in left thigh muscles.

FIG. 9: shows structures of additional CSAs.

FIG. 10: shows structures of CSA-26 and CSA-46.

FIG. 11: shows structure of CSA-134.

FIG. 12: shows structure of CSA-10.

FIG. 13: shows structure of CSA-140.

FIG. 14: shows structure of CSA-31.

FIG. 15: shows structures of CSAs 352-354.

FIG. 16: shows structures of CSAs 341-343 and 324-327.

FIG. 17: shows structure of CSA-358.

FIG. 18: shows structures of various CSAs.

DETAILED DESCRIPTION

In accordance with the invention, there are provided methods and kitsfor diagnosis, detection, screening and imaging, infection or ahyperproliferative disorder (e.g., a tumor, cancer or neoplasia, ormetastasis thereof) in vitro, ex vivo and in vivo. In one embodiment, amethod of the invention includes administering a detectably labeled CSAto the subject under conditions whereby the labeled CSA can bind to aninfection, and detecting the labeled CSA in the subject to ascertain thepresence or absence of an infection, thereby detecting the infection, ordiagnosing the subject as having or not having an infection. In anotherembodiment, a method of the invention includes administering adetectably labeled CSA to the subject under conditions whereby thelabeled CSA can bind to a tumor, cancer or neoplasia, and detecting thelabeled CSA in the subject to ascertain the presence or absence of atumor, cancer or neoplasia, thereby detecting a tumor, cancer orneoplasia, or diagnosing the subject as having or not having a tumor,cancer or neoplasia. In an additional embodiment, a method of theinvention includes administering a detectably labeled CSA to the subjectunder conditions whereby the labeled CSA can bind to an infection, andimaging the labeled CSA in the subject to ascertain the presence orabsence of an infection. In a further embodiment, a method of theinvention includes administering a detectably labeled CSA to the subjectunder conditions whereby the labeled CSA can bind to a tumor, cancer orneoplasia, and imaging the labeled CSA in the subject to ascertain thepresence or absence of a tumor, cancer or neoplasia.

The invention methods include, among other things, in vitro, ex vivo andin vivo methods. Subjects can be contacted with, administered, ordelivered a compound (e.g., one or more CSAs) in order to diagnose,detect, screen for or image an infection, or a tumor, cancer orneoplasia, or a metastasis thereof. A sample, such as a biologicalsample, can be contacted with, administered, or delivered a compound(e.g., one or more CSAs) in order to diagnose, detect, screen for orimage an infection, or a tumor, cancer or neoplasia, or a metastasisthereof.

The term “contact” and grammatical variations thereof means the subjector a sample is given or delivered a CSA under conditions allowing aphysical interaction between the CSA and an infection or ahyperproliferative disorder (e.g., a tumor, cancer or neoplasia, ormetastasis thereof) in vitro, ex vivo and in vivo. The term“administering” includes delivery to a subject in which the CSA canphysically interact with an infection or a hyperproliferative disorder(e.g., a tumor, cancer or neoplasia, or metastasis thereof) in vitro, exvivo and in vivo.

In particular embodiments of the methods and kits of the invention, aCSA is any of CSA-1 through CSA-400. In more particular aspects, a CSAis selected from: CSA-7, CSA-8, CSA-10, CSA-11, CSA-13, CSA-15, CSA-17,CSA-21, CSA-25, CSA-26, CSA-31, CSA-46, CSA-54 and CSA-59, CSA-107 andCSA-110. In other embodiments, a CSA does not have a charged group atposition C24 or a CSA has a hydrophobic moiety at position C24 (e.g., alipid). In additional embodiments, a CSA has a charged group at positionC7. In further embodiments, a CSA comprises a multimer (e.g., a dimer,trimer, tetramer or higher order polymer). In yet additionalembodiments, a CSA has a shorter tether length between the steroidscaffold and any amine group at positions C3, C7 or C12, relative to thetether length between the steroid scaffold and any amine group atpositions C3, C7 or C12 of CSA-7, CSA-8, CSA-10, CSA-11, CSA-13, CSA-15,CSA-17, CSA-21, CSA-25, CSA-26, CSA-31, CSA-46, CSA-54 or CSA-59.

Detectable labels include labels suitable for diagnosis, detection,screening or imaging. A detectable label can be included or within inthe structure of the CSA. As the structure of CSAs includes carbon,hydrogen, nitrogen, oxygen, sulfur, etc., radioisotopes of any ofcarbon, hydrogen, nitrogen, oxygen, sulfur, etc., can be included orwithin in a CSA structure such that the CSA is detectable labelled.

A detectable label can also be covalently linked or conjugated to theCSA. Non-limiting exemplary detectable labels include a radioactivematerial, such as a radioisotope, a metal or a metal oxide. Inparticular embodiments, a radioisotope can be one or more of: C, N, O,H, S, Cu, Fe, Ga, Ti, Sr, Y, Tc, In, Pm, Gd, Sm, Ho, Lu, Re, At, Bi orAc. In additional embodiments, radioisotope can be one or more of: 3H,¹⁰B, ¹⁸F, ¹¹C, ¹⁴C, ¹³N, ¹⁸O, ¹⁵O, ³²P, ³⁵S, ³⁵Cl, ⁴⁵Ti, ⁴⁶S, ⁵¹Cr,⁵²Fe, ⁵⁹Fe, ⁵⁷Co, ⁶⁰Cu, ⁶¹Cu, ⁶²Cu, ⁶⁴Cu, ⁶⁷Cu, ⁶⁷Ga, ⁶⁸Ga, ⁷²As, ⁷⁶Br,⁷⁷Br, ^(81m)Kr, ⁸²Rb, ⁸⁵Sr, ⁸⁹Sr, ⁸⁶Y, ⁹⁰Y, ⁹⁵Nb, ^(94m)Tc, ^(99m)TCl,⁹⁷Ru, ¹⁰³Ru, ¹⁰⁵Rh, ¹⁰⁹Cd, ¹¹¹In, ¹¹³Sn, ^(113m)In, ¹¹⁴In, ¹⁴⁰La, ¹⁴¹Ce¹⁴⁹Pm, ¹⁵³Gd, ¹⁵⁷Gd, ¹⁵³Sm, ¹⁶¹Tb, ¹⁶⁶Dy, ¹⁶⁶Ho, ¹⁶⁹Er, ¹⁶⁹Y, ¹⁷⁵Yb,¹⁷⁷Lu, ¹⁸⁶Re, ¹⁸⁸Re, ²⁰¹Tl, ²⁰³Pb, ²¹¹At, ²¹²Bi or ²²⁵Ac. In moreparticular embodiments, a radionuclide includes technetium and rheniumisotopes, more specifically, for example, technetium-99m, rhenium-186and rhenium-188.

Radionuclides include, but are not limited to, isotopes emitting alpha,beta or gamma radiation. In diagnostic, screening, detection and imagingmethods, typically a beta emitter is employed. Non-limiting examples ofbeta emitters include cesium-137, cobalt-60, radium-226, andtechnetium-99m.

CSAs typically include several basic sites (oxygen, sulfur, and nitrogenlone pairs) which are suitable for binding cationic radionuclides andmetals. This allows easy and optionally reversible complexation of theCSA with the radionuclide or metal. Thus, detectably labeled CSAs caninclude cationic radionuclides and cationic metals.

Additional non-limiting exemplary detectable labels include a metal ormetal oxide. In particular embodiments, a metal or metal oxide is one ormore of: gold, silver, copper, boron, manganese, gadolinium, iron,chromium, barium, europium, erbium, praseodynium, indium, or technetium.In additional embodiments, a metal oxide includes one or more of:Gd(II), Mn(II), Mn(III), Cr(II), Cr(III), Cu(II), Fe (III), Pr(III),Nd(III) Sm(III), Tb(III), Yb(III) Dy(III), Ho(III), Eu(II), Eu(III), orEr(III). Metals and oxides include crystals.

A label can also be a contrast agent (e.g., gadolinium; manganese;barium sulfate; an iodinated or noniodinated agent; an ionic agent ornonionic agent); a magnetic agent or a paramagnetic agent (e.g.,gadolinium, iron-oxide chelate); nanoparticles; an enzyme (horseradishperoxidase, alkaline phosphatase, beta.-galactosidase, oracetylcholinesterase); a prosthetic group (e.g., streptavidin/biotin andavidinfbiotin); a fluorescent material (e.g., umbelliferone,fluorescein, fluorescein isothiocyanate, rhodamine,dichlorotriazinylamine fluorescein, dansyl chloride or phycoerythrin);or a luminescent material (e.g., luminol). A label can also be abioluminescent material (e.g., luciferase, luciferin, aequorin); or anyother imaging agent that can be employed for detection, screening,diagnostic, or imaging (e.g., for CT, fluoroscopy, SPECT imaging,optical imaging, PET, MRI, gamma imaging).

The term “infection” means an initial or primary (acute) or a chronicinfection. An infection may be “infectious” in the sense that othersites in the infected host subject, or contagious to other subjects(cross-infection), or may be latent. An initial/primary (acute)infection can cause mild, moderate or severe pathogenesis or symptoms,or be asymptomatic. A primary/initial infection may or may not beself-limiting, and can become progressively worse, or become latent. A“latent” infection in a host subject is a state in which the infection(e.g., virus) evades immune clearance and remains in the host subject,which infection can be chronic, even lifelong. In the latent stateillness or symptoms may not be present or may be mild. Reactivation ofan infection means activation in the host subject following a period oflatency. Reactivation is associated with increased replication andproliferation in a subject. Symptoms and pathologies associated with orcaused by reactivation may also increase.

Specific non-limiting examples of infections include chronic, acute orlatent bacterial (gram negative and gram positive and non-gram staining)viral, parasite and fungal infections. Infections can be eitherpathogenic or non-pathogenic infections (e.g., pathogenic ornon-pathogenic bacterial, viral, parasite and fungal infections).

Specific non-limiting examples of gram negative bacterial infectionsinclude: Bordetella, Bordetella pertussis; Borrelia, Borreliaburgdorferi; Brucella, Brucella abortus, Brucella canis, Brucellamelitensis, Brucella suis, Campylobacter, Campylobacter jejuni;Escherichia, Escherichia coli; Francisella, Francisella tularensis;Haemophilus, aemophilus influenzae; Helicobacter, Helicobacter pylori;Legionella, Legionella pneumophila; Leptospira, Leptospira interrogans;Neisseria, Neisseria gonorrhoeae, Neisseria meningitides; Pseudomonas,Pseudomonas aeruginosa; Rickettsia Rickettsia rickettsii, Salmonella,Salmonella typhi, Salmonella typhimurium; Shigella Shigella sonnei;Treponema, Treponema pallidum; Vibrio, Vibrio cholerae; Yersinia,Yersinia pestis. a mycobacterium (e.g., tuberculosis and atypicalmycobacterium), listeria monocytogenes, helicobacter, bordetella,streptococcus, salmonella and chlamydia.

Specific non-limiting examples of gram positive bacterial infectionsinclude: Clostridium, Clostridium botulinum, Clostridium difficile,Clostridium perfringens, Clostridium tetani; Corynebacterium,Corynebacterium diphtheriae; Enterococcus, Enterococcus faecalis,Enterococcus faecum; Listeria Listeria, monocytogenes; Staphylococcus,Staphylococcus aureus; Staphylococcus epidermidis, Staphylococcussaprophyticus; Streptococcus, Streptococcus agalactiae; Streptococcuspneumoniae; Streptococcus pyogenes.

Specific non-limiting examples of non-gram staining bacteria include:Chlamydia, Chlamydia pneumoniae, Chlamydia psittaci, Chlamydiatrachomatis; Mycobacterium, Mycobacterium leprae, Mycobacteriumtuberculosis; Mycoplasma, Mycoplasma pneumoniae.

Specific non-limiting examples of viral infections include poxvirus,herpesvirus, hepatitis virus, immunodeficiency virus, flavivirus,papilloma virus (PV), polyoma virus, rhabdovirus, a myxovirus, anarenavirus, a coronavirus, adenovirus, reovirus, picornavirus,togavirus, bunyavirus, parvovirus or retrovirus.

Poxviruses include a vaccinia virus, Molluscum contagiosum, variolamajor smallpox virus, variola minor smallpox virus, cow pox, camel pox,sheep pox, and monkey pox. Herpesviruses include alpha-herpesvirus,beta-herpesvirus, gamma-herpesvirus, Epstein Bar Virus (EBV),Cytomegalovirus (CMV), varicella zoster virus (VZV/IHHV-3), and humanherpes simplex virus-1 (HSV-1), herpes simplex virus-2 (HSV-2) andvaricella zoster virus (VZV/HHV-3). Particular non-limiting examples ofbeta- and gamma-herpesvirus include cytomegalovirus (CMV), Epstein-Barrvirus (EBV), human herpes virus-6, -7 and -8 (HHV-6, HHV-7, orHHV-8/Kaposi's sarcoma herpesvirus/KSHV). Hepatitis viruses includehepatitis A, B, C, D, E and G. Immunodeficiency viruses include humanimmunodeficiency virus (HIV), such as HIV-1, HIV-2 and HIV-3.Flaviviruses include Hepatitis C virus, Yellow Fever virus, Denguevirus, and Japanese Encephalitis and West Nile viruses. Papillomaviruses include human papilloma virus (HPV), such as HPV strain 1, 6,11, 16, 18, 30, 31, 42, 43, 44, 45, 51, 52, and 54. Polyoma virusesinclude BK virus (BKV) and JC virus (JCV). Rhabdoviruses include rabiesvirus and vesiculovirus. Myxoviruses include paramyxovirus (e.g.,measles, mumps, pneumovirus and respiratory syncytial virus (RSV) andorthomyxovirus (e.g., influenza virus, such as influenza A, influenza Band influenza C). Arenaviruses include lymphocytic choriomeningitisvirus (LCMV), Junin virus, Lassa virus, Guanarito virus, Sabia virus andMachupo virus. Coronaviruses include viruses that cause a common cold orsevere acute respiratory syndrome (SARS). Adenoviruses include viralinfections of the bronchii, lung, stomach, intestine (gastroenteritis),eye (conjunctivitis), bladder (cystitis) and skin. Reoviruses include arotavirus, cypovirus and orbivirus. Picornaviruses include rhinovirus(e.g., causing a common cold), apthovirus, hepatovirus, enterovirus,coxsackie B virus and cardiovirus. Togaviruses include alphavirus,sindbus virus, and rubellavirus. Bunyaviruses incltide hantavirus,phlebovirus and nairovirus. Retroviruses include alpha, beta, delta,gamma, epsilon, lentivirus, spumavirus and human T-cell leukemia virus,such as human T-cell leukemia virus 1 and 2 (HTLV-1 and HTLV-2).Lentiviruses include immunodeficiency virus, such as bovine, porcine,equine, canine, feline and primate virus.

Specific non-limiting examples of parasites include a protozoa ornematode. Exemplary protozoa include a Toxoplasma gondii, Leishmania,Plasmodium, or Trypanosoma cruzi. Exemplary nematodes include aSchistosoma mansoni, or a Heligmosomoides polygyrus. Exemplary fungusincludes Candida albicans.

The terms “tumor,” “cancer” and “neoplasia” are used interchangeably andrefer to a cell or population of cells whose growth, proliferation orsurvival is greater than growth, proliferation or survival of a normalcounterpart cell, e.g. a cell proliferative, hyperproliferative ordifferentiative disorder. Typically, the growth is uncontrolled. Theterm “malignancy” refers to invasion of nearby tissue. The term“metastasis” refers to spread or dissemination of a tumor, cancer orneoplasia to other sites, locations or regions within the subject, inwhich the sites, locations or regions are distinct from the primarytumor or cancer.

Invention methods include diagnosing, detecting, screening for, orimaging a primary neoplasia, tumor cancer and metastasis thereof.Metastasis include spreading to other sites, or the formation orestablishment of neoplasia, tumors or cancers at other sites distal fromthe primary neoplasia, tumor or cancer. Thus, methods of the inventioninclude, among other things, diagnosing, detecting, screening for, orimaging a metastases arising from a primary neoplasia, tumor or cancerto one or more other sites, locations or regions distinct from theprimary neoplasia, tumor or cancer, growth or proliferation of ametastasis at one or more other sites, locations or regions distinctfrom the primary neoplasia, tumor or cancer, and formation orestablishment of additional metastasis.

Neoplasias, tumors and cancers that can be diagnosed, detected, screenedfor, or imaged include sarcoma, carcinoma, adenocarcinoma, melanoma,myeloma, blastoma, glioma, lymphoma or leukemia. Exemplary cancersinclude, for example, carcinoma, sarcoma, adenocarcinoma, melanoma,neural (blastoma, glioma), mesothelioma and reticuloendothelial,lymphatic or haematopoietic neoplastic disorders (e.g., myeloma,lymphoma or leukemia).

Neoplasia, tumors and cancers include benign, malignant, metastatic andnon-metastatic types, and include any stage (I, II, III, IV or V) orgrade (G1, G2, G3, etc.) of neoplasia, tumor, or cancer, or a neoplasia,tumor, cancer or metastasis that is progressing, worsening, stabilizedor in remission.

A “solid neoplasia, tumor or cancer” refers to neoplasia, tumor orcancer (e.g., metastasis) that typically aggregates together and forms amass. Specific examples include visceral tumors such as melanomas,breast, pancreatic, uterine and ovarian cancers, testicular cancer,including seminomas, gastric or colon cancer, hepatomas, adrenal, renaland bladder carcinomas, lung, head and neck cancers and braintumors/cancers.

Carcinomas refer to malignancies of epithelial or endocrine tissue, andinclude respiratory system carcinomas (lung, small cell lung),gastrointestinal system carcinomas, genitourinary system carcinomas,testicular carcinomas, breast carcinomas, prostatic carcinomas,endocrine system carcinomas, and melanomas. The term also includescarcinosarcomas, e.g., which include malignant tumors composed ofcarcinomatous and sarcomatous tissues. Adenocarcinoma includes acarcinoma of a glandular tissue, or in which the tumor forms a glandlike structure. Melanoma refers to malignant tumors of melanocytes andother cells derived from pigment cell origin that may arise in the skin,dermis, eye (including retina), or other regions of the body. Additionalcarcinomas can form from the uterine/cervix, endometrium, lung,head/neck, colon, pancreas, testes, adrenal gland, kidney, esophagus,stomach, liver and ovary.

Sarcomas refer to malignant tumors of mesenchymal cell origin. Exemplarysarcomas include for example, lymphosarcoma, liposarcoma, osteosarcoma,chondrosarcoma, leiomyosarcoma, rhabdomyosarcoma and fibrosarcoma.

Neural neoplasias include glioma, glioblastoma, meningioma,neuroblastoma, retinoblastoma, astrocytoma, oligodendrocytoma

Specific non-limiting examples of neoplasias, tumors and cancers includemalignant and non-malignant neoplasias, tumors and cancers, andmetastasis. In particular, a neoplasia, tumor, cancer or metastasis ofany stage (e.g., stages IA, IB, IIA, IIB, IIIA, IIIB or IV) or grade(e.g., grades G1, G2 or G3).

A “liquid neoplasia, tumor or cancer” refers to a neoplasia, tumor orcancer of the reticuloendothelial or hematopoetic system, such as alymphoma, myeloma, or leukemia, or a neoplasia that is diffuse innature. Particular examples of leukemias include acute and chroniclymphoblastic, myeolblastic and multiple myeloma. Typically, suchdiseases arise from poorly differentiated acute leukemias, e.g.,erythroblastic leukemia and acute megakaryoblastic leukemia. Specificmyeloid disorders include, but are not limited to, acute promyeloidleukemia (APML), acute myelogenous leukemia (AML) and chronicmyelogenous leukemia (CML); lymphoid malignancies include, but are notlimited to, acute lymphoblastic leukemia (ALL), which includes B-lineageALL and T-lineage ALL, chronic lymphocytic leukemia (CLL),prolymphocytic leukemia (PLL), hairy cell leukemia (HLL) andWaldenstrom's macroglobulinemia (WM). Specific malignant lymphomasinclude, non-Hodgkin lymphoma and variants, peripheral T cell lymphomas,adult T cell leukemia/lymphoma (ATL), cutaneous T-cell lymphoma (CTCL),large granular lymphocytic leukemia (LGF), Hodgkin's disease andReed-Sternberg disease.

An infection, tumor, cancer or neoplasia, or a metastasis thereof mayarise from any cause or may affect any part of the body of a subject.Exemplary parts (e.g., organ, tissue) affected include skin, dermis,breast, lung, nasopharynx, nose or sinuses, thyroid, head, neck, brain,spine, adrenal gland, thyroid, lymph, blood, gastrointestinal (mouth,esophagus, stomach, duodenum, ileum, jejunum (small intestine), colon,rectum), genito-urinary tract (uterus, ovary, endometrium, cervix,bladder, testicle, penis, urinary tract, prostate), kidney, pancreas,adrenal gland, liver, bone, bone marrow, heart, muscle, and thehematopoetic system. Thus, a method of the invention may be performed todiagnose, detect, screen, or image an infection, a tumor, cancer orneoplasia, or a metastasis thereof in the whole body of a subject, aparticular region or general area, a specific organ or tissue, or alocal portion of a region, organ or tissue.

Methods of the invention include detecting the type, kind, presence orabsence, location or extent of an infection, tumor, cancer or neoplasia,or a metastasis thereof. Such methods can be used to alternatively oradditionally provide information on severity or progression ofinfection, tumor, cancer or neoplasia, or a metastasis thereof;prognosis of infection, tumor, cancer or neoplasia, or a metastasisthereof; and/or therapy or treatment of infection, tumor, cancer orneoplasia, or a metastasis thereof based upon detecting, diagnosing,screening or imaging.

As used herein, a “sufficient amount” or “effective amount” or an“amount sufficient” or an “amount effective” refers to an amount that issufficient to detect an infection, tumor, cancer or neoplasia, or ametastasis thereof. Typically, the amount is less than an amount thatleads to substantial lysis or killing of the target infection, tumor,cancer or neoplasia, or a metastasis thereof. Thus, an amount sufficientor effective is that amount to allow detection, diagnosis, screening orimaging, without substantial cell or infection killing such that aninfection; tumor, cancer or neoplasia, or a metastasis thereof is nolonger detected, diagnosed, or imaged. Further, the amount of labeledCSA may vary with the particular label used and the method of detectionin order to achieve a desired image.

Methods of detection, diagnostics or screening, such as in vitro, exvivo, and vivo imaging methods, permit the detection of a labeled CSA.Such methods of CSA detection include magnetic resonance spectroscopy(MRS), magnetic resonance imaging (MRI), positron-emission tomography(PET), gamma-scintigraphy, computed tomography (CT), Computed AxialTomography (CAT), or single photon emission tomography (SPECT).

Methods also include detecting an infection, or diagnosing a subjecthaving or at risk of having an infection, a tumor, cancer or neoplasia,(in vivo, ex vivo or in vitro). Such methods include contacting adetectably labeled CSA to a biological sample from a subject underconditions whereby the labeled CSA can bind to an infection in thesample, and detecting the labeled CSA in the sample to ascertain thepresence or absence of an infection, a tumor, cancer or neoplasia, inthe sample, thereby of detecting an infection, a tumor, cancer orneoplasia, or diagnosing the subject as having or not having aninfection, a tumor, cancer or neoplasia.

Biological samples include any sample capable of having a biologicalmaterial. Specific non-limiting examples include mucus, saliva, feces,blood, serum, plasma, cerebrospinal fluid, urine, or placenta.Biological samples also include biopsies, for example, of skin, dermis,breast, lung, nasopharynx, nose or sinuses, thyroid, head, neck, adrenalgland, thyroid, lymph, gastrointestinal tract, genito-urinary tract,kidney, pancreas, adrenal gland, liver, bone, bone marrow, heart,muscle, or a sample of the hematopoetic system.

For in vitro, ex vivo, and in vivo diagnosing, detecting, screening orimaging, the type of detection instrument available can depend upon agiven label or conjugate. As an example, a radioisotope or paramagneticisotope is suitable for in vivo detection, diagnosis, screening orimaging. The type of lable, such as a radionuclide or metal, will guidethe selection of the instrument used. For instance, decay parameters ofa chosen alpha, beta, or gamma radionuclide chosen can be detectable ormeasured by the selected instrument.

In various embodiments a label or conjugate, such as a radionuclide ormetal or metal oxide can be bound to a CSA, either directly orindirectly, using an intermediary functional group. Intermediaryfunctional groups which are often used to bind radioisotopes which existas, for example, metallic ions (e.g, cations) that bind to groups on theCSAs. Examples include agents that react with free or semi-free amines,oxygen, sulfur, hydroxy or carboxy groups. Such functional groupstherefore include mono and bifunctional crosslinkers, such as DSS, BS3(Sulfo-DSS), DSG. Non-limiting examples includediethylenetriaminepentaacetic acid (DTPA) and ethylene diaminetetraceticacid (EDTA). Additional functional groups include carbon esideus, or oneor more of a succinly groups, such as disulfosuccinimidyl tartarate,disuccinimidyl glutarate and disuccinimidyl suberate.

A “subject” refers to an animal, typically mammalian animals, such asbut not limited to non-human primates (apes, gibbons, gorillas,chimpanzees, orangutans, macaques), domestic animals (dogs and cats), afarm animals (chickens, ducks, horses, cows, goats, sheep, pigs),experimental animal (mouse, rat, rabbit, guinea pig) and humans.Subjects include animal models, for example, a mouse model of aninfection, tumor, cancer or neoplasia, or a metastasis thereof. Subjectsinclude naturally occurring or non-naturally occurring mutated ornon-human genetically engineered (e.g., transgenic or knockout) animals.Subjects further include animals having or at risk of having aninfection, tumor, cancer or neoplasia, or a metastasis thereof. Subjectscan be any age. For example, a subject (e.g., human) can be a newborn,infant, toddler, child, teenager, or adult, e.g., 50 years or older.

Subjects include those in need of a method of the invention, e.g., inneed of diagnosis, detection, screening or imaging. A subject isconsidered to be in need of a method of the invention where a method islikely to provide information concerning the presence or absence of, theextent or severity of, the status or prognosis of, or possible treatmentor therapy of, an infection, tumor, cancer or neoplasia, or a metastasisthereof.

Subjects appropriate for treatment therefore include those having or atrisk of having an infection, tumor, cancer or neoplasia, or a metastasisthereof. At risk subjects include subjects that have been exposed to aninfection or infectious agent, or are at risk of developing a tumor,cancer or neoplasia, or a metastasis thereof, due to a geneticpredispositon or family history, or environmental risk due to smoking,exposure to smoke or carcinogens, chemicals, sun exposure, etc. Asubject may therefore be symptomatic or asymptomatic for an infection,tumor, cancer or neoplasia, or a metastasis thereof. Candidate subjectstherefore include subjects that have been exposed to or contacted withan infection, or that are at risk of exposure to or contact with aninfection, regardless of the type, timing or extent of exposure orcontact. The invention methods are therefore applicable to a subject whois at risk of an infection, tumor, cancer or neoplasia, or a metastasisthereof, but has not yet been diagnosed for an an infection, tumor,cancer or neoplasia, or a metastasis thereof. Prophylactic methods aretherefore included.

Compounds of the invention, including CSAs, can be incorporated intopharmaceutical compositions or formulations. Such pharmaceuticalcompositions/formulations are useful for administration to a subject, invivo or ex vivo.

Pharmaceutical compositions and formulations include carriers orexcipients for administration to a subject. As used herein the terms“pharmaceutically acceptable” and “physiologically acceptable” mean abiologically compatible formulation, gaseous, liquid or solid, ormixture thereof, which is suitable for one or more routes ofadministration, in vivo delivery or contact. A formulation is compatiblein that it does not destroy activity of an active ingredient therein(e.g., a CSA), or induce adverse side effects that far outweigh anyprophylactic or therapeutic effect or benefit.

Such formulations include solvents (aqueous or non-aqueous), solutions(aqueous or non-aqueous), emulsions (e.g., oil-in-water orwater-in-oil), suspensions, syrups, elixirs, dispersion and suspensionmedia, coatings, isotonic and absorption promoting or delaying agents,compatible with pharmaceutical administration or in vivo contact ordelivery. Aqueous and non-aqueous solvents, solutions and suspensionsmay include suspending agents and thickening agents. Suchpharmaceutically acceptable carriers include tablets (coated oruncoated), capsules (hard or soft), microbeads, powder, granules andcrystals. Supplementary active compounds (e.g., preservatives,antibacterial, antiviral and antifungal agents) can also be incorporatedinto the compositions.

The formulations may, for convenience, be prepared or provided as a unitdosage form. Preparation techniques include bringing into associationthe active ingredient (e.g., CSA) and a pharmaceutical carrier(s) orexcipient(s). In general, formulations are prepared by uniformly andintimately associating the active ingredient with liquid carriers orfinely divided solid carriers or both, and then, if necessary, shapingthe product. For example, a tablet may be made by compression ormolding. Compressed tablets may be prepared by compressing, in asuitable machine, an active ingredient (e.g., a CSA) in a free-flowingform such as a powder or granules, optionally mixed with a binder,lubricant, inert diluent, preservative, surface-active or dispersingagent. Molded tablets may be produced by molding, in a suitableapparatus, a mixture of powdered compound (e.g., CSA) moistened with aninert liquid diluent. The tablets may optionally be coated or scored andmay be formulated so as to provide a slow or controlled release of theactive ingredient therein.

Pharmaceutical compositions can optionally be formulated to becompatible with a particular route of administration. Exemplary routesof administration include administration to a biological fluid ortissue, mucosal cell or tissue (e.g., mouth, buccal cavity, labia,nasopharynx, esophagus, trachea, lung, stomach, small intestine, vagina,rectum, or colon), neural cell or tissue (e.g., ganglia, motor orsensory neurons) or epithelial cell or tissue (e.g., nose, fingers,ears, cornea, conjunctiva, skin or dermis). Thus, pharmaceuticalcompositions include carriers (excipients, diluents, vehicles or fillingagents) suitable for administration to any cell, tissue or organ, invivo, ex vivo (e.g., tissue or organ transplant) or in vitro, by variousroutes and delivery, locally, regionally or systemically.

Exemplary routes of administration for contact or in vivo delivery whicha compound of the invention (e.g., CSA) can optionally be formulatedinclude inhalation, respiration, intubation, intrapulmonaryinstillation, oral (buccal, sublingual, mucosal), intrapulmonary,rectal, vaginal, intrauterine, intradermal, topical, dermal, parenteral(e.g., subcutaneous, intramuscular, intravenous, intradermal,intraocular, intratracheal and epidural), intranasal, intrathecal,intraarticular, intracavity, transdermal, iontophoretic, ophthalmic,optical (e.g., corneal), intraglandular, intraorgan, intralymphatic.

Formulations suitable for parenteral administration include aqueous andnon-aqueous solutions, suspensions or emulsions of the compound, whichmay include suspending agents and thickening agents, which preparationsare typically sterile and can be isotonic with the blood of the intendedrecipient. Non-limiting illustrative examples of aqueous carriersinclude water, saline (sodium chloride solution), dextrose (e.g.,Ringer's dextrose), lactated Ringer's, fructose, ethanol, animal,vegetable or synthetic oils. Examples of non-aqueous solvents arepropylene glycol, polyethylene glycol, vegetable oils such as olive oil,and injectable organic esters such as ethyl oleate. Intravenous vehiclesinclude fluid and nutrient replenishers, electrolyte replenishers (suchas those based on Ringer's dextrose). The formulations may be presentedin unit-dose or multi-dose kits, for example, ampules and vials, and maybe stored in a freeze-dried (lyophilized) condition requiring additionof a sterile liquid carrier, for example, water for injections, prior touse.

For transmucosal or transdermal administration (e.g., topical contact),penetrants can be included in the pharmaceutical composition. Penetrantsare known in the art, and include, for example, for transmucosaladministration, detergents, bile salts, and fusidic acid derivatives.For transdermal administration, the active ingredient can be formulatedinto aerosols, sprays, ointments, salves, gels, pastes, lotions, oils orcreams as generally known in the art.

For topical administration, for example, to skin, pharmaceuticalcompositions typically include ointments, creams, lotions, pastes, gels,sprays, aerosols or oils. Carriers which may be used include Vaseline,lanolin, polyethylene glycols, alcohols, transdermal enhancers, andcombinations thereof.

For oral administration, pharmaceutical compositions include capsules,cachets, lozenges, tablets or troches, as powder or granules. Oraladministration formulations also include a solution or a suspension(e.g., aqueous liquid or a non-aqueous liquid; or as an oil-in-waterliquid emulsion or a water-in-oil emulsion).

For airway or nasal administration, pharmaceutical compositions can beformulated in a dry powder for delivery, such as a fine or a coarsepowder having a particle size, for example, in the range of 20 to 500microns which is administered in the manner by inhalation through theairways or nasal passage. Depending on delivery device efficiency,effective dry powder dosage levels typically fall in the range of about10 to about 100 mg. Appropriate formulations, wherein the carrier is aliquid, for administration, as for example, a nasal spray or as nasaldrops, include aqueous or oily solutions of the active ingredient.Dry-powder inhalers (DPI) can be used to deliver the compounds (CSAs),either alone or in combination with a pharmaceutically acceptablecarrier.

For airway or nasal administration, aerosol and spray delivery systemsand devices, also referred to as “aerosol generators” and “spraygenerators,” such as metered dose inhalers (MDI), nebulizers(ultrasonic, electronic and other nebulizers), nasal sprayers and drypowder inhalers can be used. MDIs typically include an actuator, ametering valve, and a container that holds a suspension or solution,propellant, and surfactant (e.g., oleic acid, sorbitan trioleate,lecithin). Activation of the actuator causes a predetermined amount tobe dispensed from the container in the form of an aerosol, which isinhaled by the subject.

For rectal administration, pharmaceutical compositions can be includedas a suppository with a suitable base comprising, for example, cocoabutter or a salicylate. For vaginal administration, pharmaceuticalcompositions can be included as pessaries, tampons, creams, gels,pastes, foams or spray formulations containing in addition to the activeingredient (e.g., CSA) a carrier, examples of appropriate carriers whichare known in the art.

Pharmaceutical formulations and delivery systems appropriate for thecompositions and methods of the invention are known in the art (see,e.g., Remington: The Science and Practice of Pharmacy (2003) 20^(th)ed., Mack Publishing Co., Easton, Pa.; Remington's PharmaceuticalSciences (1990) 18^(th) ed., Mack Publishing Co., Easton, Pa.; The MerckIndex (1996) 12^(th) ed., Merck Publishing Group, Whitehouse, NJ;Pharmaceutical Principles of Solid Dosage Forms (1993), TechnonicPublishing Co., Inc., Lancaster, Pa.; Ansel and Stoklosa, PharmaceuticalCalculations (2001) 11^(th) ed., Lippincott Williams & Wilkins,Baltimore, Md.; and Poznansky et al., Drug Delivery Systems (1980), R.L. Juliano, ed., Oxford, N.Y., pp. 253-315).

Compounds of the invention (e.g., CSAs), including pharmaceuticalformulations can be packaged in unit dosage forms for ease ofadministration and uniformity of dosage. A “unit dosage form” as usedherein refers to a physically discrete unit suited as unitary dosagesfor the subject to be administered or contacted; each unit containing apredetermined quantity of compound optionally in association with apharmaceutical carrier (excipient, diluent, vehicle or filling agent).Unit dosage forms can contain a daily dose or unit, daily sub-dose, oran appropriate fraction thereof, of an administered compound (e.g.,CSA). Unit dosage forms also include, for example, capsules, troches,cachets, lozenges, tablets, ampules and vials, which may include acomposition in a freeze-dried or lyophilized state; a sterile liquidcarrier, for example, can be added prior to administration, contact, ordelivery in vivo. Unit dosage forms additionally include, for example,ampules and vials with liquid compositions disposed therein. Unit dosageforms further include compounds for transdermal administration, such as“patches” that contact with the epidermis of the subject for an extendedor brief period of time. The individual unit dosage forms can beincluded in multi-dose kits or containers. Pharmaceutical formulationscan be packaged in single or multiple unit dosage forms for ease ofadministration and uniformity of dosage.

Compounds of the invention (e.g., CSAs) can be administered at anyduration or frequency. Typically, a labeled CSA is administered as abolus or is administered in multiple dose to provide detection,diagnosis, screening or imaging.

Exemplary non-limiting doses include, for example, those based on themass of a subject. Doses can generally be in a range from about 0.1-1ug/kg, 1-10 ug/kg, 10-25 ug/kg, 25-50 ug/kg, 50-100 ug/kg, 100-500ug/kg, 500-1,000 ug/kg, 1-4 mg/kg, 4-10 mg/kg, 10-20 mg/kg, 20-50 mg/kg,50-100 mg/kg, 100-250 mg/kg, 250-500 mg/kg, or more, of subject bodyweight, two, three, four, or more times per hour, day, week, month orannually. Of course, doses can be more or less, as appropriate, forexample, 0.00001 mg/kg of subject body weight to about 10,000.0 mg/kg ofsubject body weight, about 0.001 mg/kg, to about 100 mg/kg, about 0.01mg/kg, to about 10 mg/kg, or about 0.1 mg/kg, to about 1 mg/kg ofsubject body weight over a given time period, e.g., 1, 2, 3, 4, 5 ormore hours, days, weeks, months, years. A subject may be administeredsingle bolus or in divided/metered doses, which can be adjusted to bemore or less according to the various consideration set forth herein andknown in the art. Dosage levels of labeled CSA also can take intoconsideration the particular detectable label and detection system inorder to achieve a desired image.

The invention provides kits including compounds of the invention (e.g.,CSA), combination compositions and pharmaceuticalcompositions/formulations thereof, packaged into a suitable packagingmaterial. In one embodiment, a kit includes packaging material, acationic steroid antimicrobial (CSA) and instructions. In variousaspects, the instructions are for administering the CSA to diagnose,detect, screen or image an infection, tumor, cancer or neoplasia, or ametastasis thereof.

The term “packaging material” refers to a physical structure housing oneor more components of the kit. The packaging material can maintain thecomponents sterilely, and can be made of material commonly used for suchpurposes (e.g., paper, corrugated fiber, glass, plastic, foil, ampules,vials, tubes, etc.). A kit can contain a plurality of components, e.g.,two or more CSAs of the invention alone or in combination.

A kit optionally includes a label or insert including a description ofthe components (type, amounts, doses, etc.), instructions for use invitro, in vivo, or ex vivo, and any other components therein. Labels orinserts include “printed matter,” e.g., paper or cardboard, or separateor affixed to a component, a kit or packing material (e.g., a box), orattached to an ampule, tube or vial containing a kit component. Labelsor inserts can additionally include a computer readable medium, such asa disk (e.g., hard disk, ZIP disk), optical disk such as CD- orDVD-ROM/RAM, DVD, MP3, magnetic tape, or an electrical storage mediasuch as RAM and ROM or hybrids of these such as magnetic/optical storagemedia, FLASH media or memory type cards.

Labels or inserts can include identifying information of one or morecomponents therein, dose amounts, clinical pharmacology of the activeingredient(s) including mechanism of action, pharmacokinetics andpharmacodynamics. Labels or inserts can include information identifyingmanufacturer, lot numbers, manufacturer location and date, expirationdates.

Labels or inserts can include information on a infection, disorder ordisease (e.g., bacterial, virus infection, tumor, neoplasia or cancer)for which a kit component may be used. Labels or inserts can includeinstructions for a clinician or subject for using one or more of the kitcomponents in a method, treatment protocol or therapeutic/prophylacticregimen, including the methods of the invention. Instructions caninclude amounts of compound, frequency or duration of administration,and instructions for practicing any of the methods, treatment protocolsor prophylactic or therapeutic regimes described herein. Kits thereforecan additionally include labels or instructions for practicing any ofthe methods of the invention described herein including detection,diagnosis, screening or other methods.

Invention kits can additionally include a buffering agent, or apreservative or a stabilizing agent in a pharmaceutical formulationcontaining a compound of the invention. Each component of the kit can beenclosed within an individual container and all of the variouscontainers can be within a single package. Invention kits can bedesigned for cold storage.

Compounds useful in accordance with the invention, are described herein,both generically and with particularity, and in U.S. Pat. Nos.6,350,738; 6,486,148; and 6,767,904, which are incorporated herein byreference in their entirety. Compounds include steroid derivatives, suchas cationic steroid antimicrobials (CSA). The skilled artisan willrecognize the compounds within the generic formula set forth herein.Particular CSAs are described herein and can be characterized using theassays set forth herein and in the art.

Compounds of formula I, also referred to as cationic steroidantimicrobials (CSA), comprise:

wherein:fused rings A, B, C, and D are independently saturated or fully orpartially unsaturated; andeach of R₁ through R₄, R₆, R₇, R₁₁, R₁₂, R₁₅, R₁₆, and R₁₇ isindependently selected from the group consisting of hydrogen, hydroxyl,a substituted or unsubstituted (C1-C10) alkyl, (C1-C10) hydroxyalkyl,(C1-C10) alkyloxy-(C1-C10) alkyl, (C1-C10) alkylcarboxy-(C1-C10) alkyl,(C1-C10) alkylamino-(C1-C10) alkyl, (C1-C10) alkylamino-(C1-C10)alkylamino, (C1-C10) alkylamino-(C1-C10) alkylamino-(C1-C10) alkylamino,a substituted or unsubstituted (C1-C10) aminoalkyl, a substituted orunsubstituted aryl, a substituted or unsubstituted arylamino-(C1-C10)alkyl, (C1-C10) haloalkyl, C2-C6 alkenyl, C2-C6 alkynyl, oxo, a linkinggroup attached to a second steroid, a substituted or unsubstituted(C1-C10) aminoalkyloxy, a substituted or unsubstituted (C1-C10)aminoalkyloxy-(C1-C10) alkyl, a substituted or unsubstituted (C1-C10)aminoalkylcarboxy, a substituted or unsubstituted (C1-C10)aminoalkylaminocarbonyl, a substituted or unsubstituted (C1-C10)aminoalkylcarboxamido, H₂N—HC(Q5)-C(O)—O—, H2N—HC(Q5)-C(O)—N(H)—,(C1-C10) azidoalkyloxy, (C1-C10) cyanoalkyloxy, P.G.-HN—HC(Q5)-C(O)—O—,(C1-C10) guanidinoalkyloxy, (C1-C10) quaternaryammoniumalkylcarboxy, and(C1-C10) guanidinoalkyl carboxy, where Q5 is a side chain of any aminoacid (including the side chain of glycine, i.e., H), P.G. is an aminoprotecting group, and

R₅, R₈, R₉, R₁₀, R₁₃, and R₁₄ is each independently: deleted when one offused rings A, B, C, or D is unsaturated so as to complete the valencyof the carbon atom at that site, or selected from the group consistingof hydrogen, hydroxyl, a substituted or unsubstituted (C1-C10) alkyl,(C1-C10) hydroxyalkyl, (C1-C10) alkyloxy-(C1-C10) alkyl, a substitutedor unsubstituted (C1-C10) aminoalkyl, a substituted or unsubstitutedaryl, C1-C10 haloalkyl, C2-C6 alkenyl, C2-C6 alkynyl, oxo, a linkinggroup attached to a second steroid, a substituted or unsubstituted(C1-C10) aminoalkyloxy, a substituted or unsubstituted (C1-C10)aminoalkylcarboxy, a substituted or unsubstituted (C1-C10)aminoalkylaminocarbonyl, H2N—HC(Q5)-C(O)—O—, H2N—HC(Q5)-C(O)—N(H)—,(C1-C10) azidoalkyloxy, (C1-C10) cyanoalkyloxy, P.G.-HN—HC(Q5)-C(O)—O—,(C1-C10) guanidinoalkyloxy, and (C1-C10) guanidinoalkylcarboxy, where Q5is a side chain of any amino acid, P.G. is an amino protecting group,and provided that at least two of R₁ through R₁₄ are independentlyselected from the group consisting of a substituted or unsubstituted(C1-C10) aminoalkyloxy, (C1-C10) alkylcarboxy-(C1-C10) alkyl, (C1-C10)alkylamino-(C1-C10) alkylamino, (C1-C10) alkylamino-(C1-C10)alkylamino-(C1-C10) alkylamino, a substituted or unsubstituted (C1-C10)aminoalkylcarboxy, a substituted or unsubstituted arylamino-(C1-C10)alkyl, a substituted or unsubstituted (C1-C10) aminoalkyloxy-(C1-C10)alkyl, a substituted or unsubstituted (C1-C10) aminoalkylaminocarbonyl,(C1-C10) quaternaryammonium alkylcarboxy, H2N—HC(Q5)-C(O)—O—,H2N—HC(Q5)-C(O)—N(H)—, (C1-C10) azidoalkyloxy, (C1-C10) cyanoalkyloxy,P.G.-HN—HC(Q5)-C(O)—O—, (C1-C10) guanidinoalkyloxy, and (C1-C10)guanidinoalkylcarboxy; or a pharmaceutically acceptable salt thereof.

A “ring” as used herein can be heterocyclic or carbocyclic. The term“saturated” used herein refers to the fused ring of formula I havingeach atom in the fused ring either hydrogenated or substituted such thatthe valency of each atom is filled. The term “unsaturated” used hereinrefers to the fused ring of formula I where the valency of each atom ofthe fused ring may not be filled with hydrogen or other substituents.For example, adjacent carbon atoms in the fused ring can be doubly boundto each other. Unsaturation can also include deleting at least one ofthe following pairs and completing the valency of the ring carbon atomsat these deleted positions with a double bond; such as R₅ and R₉; R₈ andR₁₀; and R₁₃ and R₁₄.

The term “unsubstituted” used herein refers to a moiety having each atomhydrogenated such that the valency of each atom is filled.

The term “halo” used herein refers to a halogen atom such as fluorine,chlorine, bromine, or iodine.

Examples of amino acid side chains include but are not limited to H(glycine), methyl (alanine), —CH₂—(C═O)—NH₂ (asparagine), —CH₂—SH(cysteine), and —CH(OH)CH₃ (threonine).

An alkyl group is a branched or unbranched hydrocarbon that may besubstituted or unsubstituted. Examples of branched alkyl groups includeisopropyl, sec-butyl, isobutyl, tert-butyl, sec-pentyl, isopentyl,tert-pentyl, isohexyl. Substituted alkyl groups may have one, two, threeor more substituents, which may be the same or different, each replacinga hydrogen atom. Substituents are halogen (e.g., F, Cl, Br, and I),hydroxyl, protected hydroxyl, amino, protected amino, carboxy, protectedcarboxy, cyano, methylsulfonylamino, alkoxy, acyloxy, nitro, and lowerhaloalkyl.

The term “substituted” used herein refers to moieties having one, two,three or more substituents, which may be the same or different, eachreplacing a hydrogen atom. Examples of substituents include but are notlimited to halogen (e.g., F, Cl, Br, and I), hydroxyl, protectedhydroxyl, amino, protected amino, carboxy, protected carboxy, cyano,methylsulfonylamino, alkoxy, alkyl, aryl, aralkyl, acyloxy, nitro, andlower haloalkyl.

An aryl group is a C6-20 aromatic ring, wherein the ring is made ofcarbon atoms (e.g., C6-C14, C6-10 aryl groups). Examples of haloalkylinclude fluoromethyl, dichloromethyl, trifluoromethyl,1,1-difluoroethyl, and 2,2-dibromoethyl.

An aralkyl group is a group containing 6-20 carbon atoms that has atleast one aryl ring and at least one alkyl or alkylene chain connectedto that ring. An example of an aralkyl group is a benzyl group.

A linking group is any divalent moiety used to link a compound offormula to another steroid, e.g., a second compound of formula I. Anexample of a linking group is (C1-C10) alkyloxy-(C1-C10) alkyl.

Amino-protecting groups are known to those skilled in the art. Ingeneral, the species of protecting group is not critical, provided thatit is stable to the conditions of any subsequent reaction(s) on otherpositions of the compound and can be removed at the appropriate pointwithout adversely affecting the remainder of the molecule. In addition,a protecting group may be substituted for another after substantivesynthetic transformations are complete. Clearly, where a compounddiffers from a compound disclosed herein only in that one or moreprotecting groups of the disclosed compound has been substituted with adifferent protecting group, that compound is within the invention.Further examples and conditions are found in T. W. Greene, ProtectiveGroups in Organic Chemistry, (1st ed., 1981, 2nd ed., 1991).

The invention compounds also include a ring system of at least 4 fusedrings, where each of the rings has from 5-7 atoms. The ring system hastwo faces, and contains 3 chains attached to the same face. Each of thechains contains a nitrogen-containing group that is separated from thering system by at least one atom; the nitrogen-containing group is anamino group, e.g., a primary amino group, or a guanidino group. Thecompound can also contain a hydrophobic group, such as a substituted(C3-10) aminoalkyl group, a (C1-10) alkyloxy (C3-10) alkyl group, or a(C1-10) alkylamino (C3-10)alkyl group, attached to the steroid backbone.For example, the compound may have the formula V, where each of thethree chains containing nitrogen-containing groups is independentlyselected from R₁ through R₄, R₆, R₇, R₁₁, R₁₂, R₁₅, R₁₆, R₁₇, and R₁₈,defined below.

where:each of fused rings A, B, C, and D is independently saturated, or isfully or partially unsaturated, provided that at least two of A, B, C,and D are saturated, wherein rings A, B, C, and D form a ring system;each of m, n, p, and q is independently 0 or 1;each of R₁ through R₄, R₆, R₇, R₁₁, R₁₂, R₁₅, R₁₆, R₁₇, and R₁₈ isindependently selected from the group consisting of hydrogen, hydroxyl,a substituted or unsubstituted (C1-C10) alkyl, (C1-C10) hydroxyalkyl,(C1-C10) alkyloxy-(C1-C10) alkyl, (C1-C10)alkylcarboxy-(C1-C10 alkyl,(C1-C10) alkylamino-(C1-C10) alkyl, (C1-C10) alkylamino-(C1-C10)alkylamino, (C1-C10 alkylamino-(C1-C10) alkylamino-(C1-C10) alkylamino,a substituted or unsubstituted (C1-C10) aminoalkyl, a substituted orunsubstituted aryl, a substituted or unsubstituted arylamino-(C1-C10)alkyl, (C1-C10) haloalkyl, C2-C6 alkenyl, C2-C6 alkynyl, oxo, a linkinggroup attached to a second steroid, a substituted or unsubstituted(C1-C10) aminoalkyloxy, a substituted or unsubstituted (C1-C10)aminoalkyloxy-(C1-C10) alkyl, a substituted or unsubstituted (C1-C10)aminoalkylcarboxy, a substituted or unsubstituted (C1-C10)aminoalkylaminocarbonyl, a substituted or unsubstituted (C1-C10)aminoalkylcarboxamido, H₂N—HC(Q5)-C(O)—O—, H2N—HC(Q5)-C(O)—N(H)—,(C1-C10) azidoalkyloxy, (C1-C10) cyanoalkyloxy, P.G.-HN—HC(Q5)-C(O)—O—,(C1-C10) guanidinoalkyl oxy, (C1-C10) quaternaryammoniumalkylcarboxy,and (C1-C10) guanidinoalkyl carboxy, where Q5 is a side chain of anyamino acid (including a side chain of glycine, i.e., H). P.G. is anamino protecting group: andeach of R₅, R₈, R₉, R₁₀, R₁₃, and R₁₄ is independently: deleted when oneof fused rings A, B, C, or D is unsaturated so as to complete thevalency of the carbon atom at that site, or selected from the groupconsisting of hydrogen, hydroxyl, a substituted or unsubstituted(C1-C10) alkyl, (C1-C10) hydroxyalkyl, (C1-C10) alkyloxy-(C1-C10) alkyl,a substituted or unsubstituted (C1-C10) aminoalkyl, a substituted orunsubstituted aryl, C1-C10 haloalkyl, C2-C6 alkenyl, C2-C6 alkynyl, oxo,a linking group attached to a second steroid, a substituted orunsubstituted (C1-C10) aminoalkyloxy, a substituted or unsubstituted(C1-C10) aminoalkylcarboxy, a substituted or unsubstituted (C1-C10)aminoalkylaminocarbonyl, H2N—HC(Q5)-C(O)—O—, H2N—HC(Q5)-C(O)—N(H)—,(C1-C10) azidoalkyloxy, (C1-C10) cyanoalkyloxy, P.G.-HN—HC(Q5)-C(O)—O—,(C1-C10) guanidinoalkyloxy, and (C1-C10) guanidinoalkylcarboxy, where Q5is a side chain of any amino acid, P.G. is an amino protecting group,provided that at least three of R₁ through R₄, R₆, R₇, R₁₁, R₁₂, R₁₅,R₁₆, R₁₇, and R₁₈ are disposed on the same face of the ring system andare independently selected from the group consisting of a substituted orunsubstituted (C1-C10) aminoalkyl, a substituted or unsubstituted(C1-C10) aminoalkyloxy, (C1-C10) alkylcarboxy-(C1-C10) alkyl, (C1-C10)alkylamino-(C1-C10) alkylamino, (C1-C10) alkylamino-(C1-C10)alkylamino-(C1-C10) alkylamino, a substituted or unsubstituted (C1-C10)aminoalkylcarboxy, a substituted or unsubstituted arylamino-(C1-C10)alkyl, a substituted or unsubstituted (C1-C10) aminoalkyloxy-(C1-C10)aminoalkylaninocarbonyl, a substituted or unsubstituted (C1-C10)aminoalkylaniinocarbonyl, a substituted or unsubstituted (C1-C5)aminoalkylcarboxamido, a (C1-C10) quatemaryammoniumalkylcarboxy,H2N—HC(Q5)-C(O)—O—, H2N—HC(Q5)-C(O)—N(H)—, (C1-C10) azidoalkyloxy,(C1-C10) cyanoalkylox, P.G.-HN—HC(Q5)-C(O)—O—, (C1-C10)guanidinoalkyloxy, and a (C1-C10) guanidinoalkylcarboxy; or apharmaceutically acceptable salt thereof. In various aspects, at leasttwo, or at least, three, of m, n, p, and q are 1.

Compounds set forth herein preserve certain stereochemical andelectronic characteristics found in steroids. The term “sameconfiguration” as used herein refers to substituents on the fusedsteroid having the same stereochemical orientation. For examplesubstituents R₃, R₇ and R₁₂ are all 1-substituted or α-substituted.

Compounds include but are not limited to compounds having amine orguanidine groups covalently attached to a steroid backbone or scaffoldat any carbon position, e.g., cholic acid. In various embodiments, agroup is covalently attached at any one, or more, of positions C3, C7and C12 of the steroid backbone or scaffold. In additional embodiments,a group is absent from any one, or more, of positions C3, C7 and C12 ofthe steroid backbone or scaffold.

Compounds that include such groups can include a tether, the tetherhaving variable chain length or size. As used herein, the terms “tether”or “tethered,” when used in reference to a compound of the invention,refers to the chain of atoms between the steroid backbone or scaffoldand a terminal amino or guanidine group. In various embodiments, atether is covalently attached at any one, or more, of positions C3, C7and C12. In additional embodiments, a tether is lacking at any one, ormore, of positions C3, C7 and C12. A tether length may include theheteroatom (O or N) covalently attached to the steroid backbone.

Other ring systems can also be used, e.g., 5-member fused rings.Compounds with backbones having a combination of 5- and 6-membered ringsare also included in the invention. Amine or guanidine groups can beseparated from the backbone by at least one, two, three, four or moreatoms. The backbone can be used to orient the amine or guanidine groupson one face, or plane, of the steroid. For example, a scheme showing acompound having primary amino groups on one face, or plane, of abackbone is shown below:

Methods of synthesizing compounds of formula I are provided, wherein forexample, at least two of R₁ through R₁₄ are independently selected fromthe group consisting of a substituted or unsubstituted (C1-C10)aminoalkyloxy. In one embodiment, a method includes the step ofcontacting a compound of formula IV,

where at least two of R₁ through R₁₄ are hydroxyl, and the remainingmoieties on the fused rings A, B, C, and D are defined for formula I,with an electrophile to produce an alkyl ether compound of formula IV,wherein at least two of R₁ through R₁₄ are (C1-C10)alkyloxy. The alkylether compounds are converted into an amino precursor compound whereinat least two of R₁ through R₁₄ are independently selected from the groupconsisting of (C1-C10) azidoalkyloxy and (C1-C10) cyanoalkyloxy and theamino precursor compound is reduced to form a compound of formula I.

Electrophiles include but are not limited to2-(2-bromoethyl)-1,3-dioxolane, 2-iodoacetamide, 2-chloroacetamide,N-(2-bromoethyl)phthalimide, N-(3-bromopropyl)phthalimide, andallybromide. An exemplary electrophile is allylbromide.

Compounds of formula I include at least two of R₁ through R₁₄ are(C1-C10) guanidoalkyloxy. In one embodiment, a method includescontacting a compound of formula IV, where at least two of R₁ throughR₁₄ are hydroxyl, with an electrophile to produce an alkyl ethercompound of formula IV, where at least two of R₁ through R₁₄ are(C1-C10)alkyloxy. The allyl ether compound is converted into an aminoprecursor compound where at least two of R₁ through R₁₄ areindependently selected from the group consisting of (C1-C10)azidoalkyloxy and (C1-C10) cyanoalkyloxy. The amino precursor compoundis reduced to produce an aminoalkyl ether compound wherein at least twoof R₁ through R₁₄ are (C1-C10) aminoalkyloxy. The aminoalkyl ethercompound is contacted with a guanidino producing electrophile to form acompound of formula I.

The term “guanidino producing electrophile” used herein refers to anelectrophile used to produce a guanidino compound of formula I. Anexample of an guanidino producing electrophile is HSO₃—C(NH)—NH₂.

Compounds of formula I also include at least two of R₁ through R₁₄ areH2N—HC(Q5)-C(O)—O— and Q5 is the side chain of any amino acid. In oneembodiment, a method includes the step of contacting a compound offormula IV, where at least two of R, through R₁₄ are hydroxyl, with aprotected amino acid to produce a protected amino acid compound offormula IV where at least two of at least two of R₁ through R₁₄ areP.G.-HN—HC(Q5)-C(O)—O— and Q5 is the side chain of any amino acid andP.G. is an amino protecting group. The protecting group of the protectedamino acid compound is removed to form a compound of formula I.

Exemplary non-limiting synthesis schemes for preparing compounds of theinvention include the following:

Compounds of the invention and precursors to the compounds according tothe invention are available commercially, e.g., from Sigm-Aldrich Co.,St. Louis; MO; and Research Plus, Inc., Manasquan, N.J. Other compoundsaccording to the invention can be synthesized according to methodsdiosclosed herein, in U.S. Pat. Nos. 6,350,738; 6,486,148; and6,767,904, and in the art.

Unless otherwise defined, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. Although methods and materialssimilar or equivalent to those described herein can be used in thepractice or study of the present invention, suitable methods andmaterials are described herein.

All of the features disclosed herein may be combined in any combination.Each feature disclosed in the specification may be replaced by analternative feature serving a same, equivalent, or similar purpose.Thus, unless expressly stated otherwise, disclosed features (e.g.,compound structures) are an example of a genus of equivalent or similarfeatures.

All applications, publications, patents and other references, GenBankcitations and ATCC citations cited herein are incorporated by referencein their entirety. In case of conflict, the specification, includingdefinitions, will control.

As used herein, the singular forms “a”, “and,” and “the” include pluralreferents unless the context clearly indicates otherwise. Thus, forexample, reference to “a compound” or “a CSA” includes a plurality ofcompounds/CSAs and reference to “an infection” can include reference toone or more infections, and so forth.

As used herein, all numerical values or numerical ranges includeintegers within such ranges and fractions of the values or the integerswithin ranges unless the context clearly indicates otherwise. Thus, toillustrate, reference to a range of 90-100%, includes 91%, 92%, 93%,94%, 95%, 95%, 97%, etc., as well as 91.1%, 91.2%, 91.3%, 91.4%, 91.5%,etc., 92.1%, 92.2%, 92.3%, 92.4%, 92.5%, etc., and so forth. Referenceto a range of 0-72 hrs, includes 1, 2, 3, 4, 5, 6, 7 hrs, etc., as wellas 1, 2, 3, 4, 5, 6, 7 minutes, etc., and so forth. Reference to a rangeof 0-72 hrs, includes 1, 2, 3, 4, 5, 6, 7 hrs, etc., as well as 1, 2, 3,4, 5, 6, 7 minutes, etc., and so forth. Reference to a range of doses,such as 0.1-1 ug/kg, 1-10 ug/kg, 10-25 ug/kg, 25-50 ug/kg, 50-100 ug/kg,100-500 ug/kg, 500-1,000 ug/kg, 1-5 mg/kg, 5-10 mg/kg, 10-20 mg/kg,20-50 mg/kg, 50-100 mg/kg, 100-250 mg/kg, 250-500 mg/kg, includes0.11-0.9 ug/kg, 2-9 ug/kg, 11.5-24.5 ug/kg, 26-49 ug/kg, 55-90 ug/kg,125-400 ug/kg, 750-800 ug/kg, 1.1-4.9 mg/kg, 6-9 mg/kg, 11.5-19.5 mg/kg,21-49 mg/kg, 55-90 mg/kg, 125-200 mg/kg, 275.5-450.1 mg/kg, etc. Aseries of ranges, for example, 1-10 ug/kg, 10-25 ug/kg, 25-50 ug/kg,50-100 ug/kg, 100-500 ug/kg, 500-1,000 ug/kg, 1-5 mg/kg, 5-10 mg/kg,10-20 mg/kg, 20-50 mg/kg, 50-100 mg/kg, 100-250 mg/kg, 250-500 mg/kg,includes 1-25 ug/kg, 10-25 ug/kg, 25-100 ug/kg, 100-1,000 ug/kg, 1-10mg/kg, 1-20 mg/kg etc.

The invention is generally disclosed herein using affirmative languageto describe the numerous embodiments. The invention also includesembodiments in which subject matter is excluded, in full or in part,such as substances or materials, method steps and conditions, protocols,or procedures. Thus, even though the invention is generally notexpressed herein in terms of what the invention does not include aspectsthat are not expressly excluded in the invention are neverthelessdisclosed herein.

A number of embodiments of the invention have been described.Nevertheless, one skilled in the art, without departing from the spiritand scope of the invention, can make various changes and modificationsof the invention to adapt it to various usages and conditions. Forexample, salts, esters, ethers and amides of invention compoundsdisclosed herein are within the scope of this invention.

Accordingly, the following examples are intended to illustrate but notlimit the scope of invention described in the claims.

EXAMPLES

CSA compounds and intermediates were characterized using the followinginstruments: ¹H and ¹³C NMR spectra were recorded on a Varian Gemini2000 (200 MHz), Varian Unity 300 (300 MHz), or Varian VXR 500 (500 MHz)spectrometer and are referenced to TMS, residual CHCl₃ (¹H) or CDCl₃(¹³C), or residual CHD₂OD (¹H), or CD₃OD (¹³C). IR spectra were recordedon a Perkin Elmer 1600 FTIR instrument. Mass spectrometric data wereobtained on a JOEL SX 102A spectrometer. THF solvent was dried overNalbenzophenone and CH₂Cl₂ was dried over CaH₂ prior to use. Otherreagents and solvents were obtained commercially and were used asreceived.

Example 1

This example includes a description of various Materials and Methods.

CSA-13 was obtained from the Paul B. Savage laboratory, Department ofChemistry and Biochemistry, Brigham Young University, C100 BNSN, Provo,Utah 84602. Rats (Sprague-Dawley) and staphylococcus aureus bacteria(American type culture collection, ATCC 25923) were obtained from theNational Institute of Health (NIH) Islamabad. The Animal ethicsCommittee of Institute gave an ethical approval for the animal studies.^(99m)Tc was obtained from locally produced fission based PAKGEN⁹⁹Mo/^(99m)Tc generator system. All the chemicals used were AR grade andpurchased from Merck., Germany. Freeze dried kits of DTPA andCiprofloxacin were obtained from “Kit Production Group”, PINSTECH,Islamabad.

Radiolabeling of DTPA, Ciprofloxacin and CSA-13

DTPA kits contain 10 mg of diethylene triamine pentaacetic acid, 0.4 mgof SnCl₂.2H₂O and NaOH to make pH 7. 1.0 ml of Na^(99m)TcO₄ (8-10 mCi)was then added and incubated at room temperature for 20 min.Ciprofloxacin kits contain 3.8 mg of ciprofloxacin, 0.39 mg ofSnCl₂.2H₂O, 4.4 mg of NaCl and 6 ml of acetate buffer pH 3.4 in 4.0 mlof H₂O, prepared as described (Obradovic, V., et al., World J Nuc. Med2:269 (2003)). 2.0 ml of Na^(99m)TcO₄ (8-10 mCi) was then added andincubated at room temperature for 10 min. Stock solution of CSA-13 (2mg/1 ml) was prepared in distilled water and 0.1 ml aliquots (˜200 μg)of CSA-13 were stored at 4° C. CSA-13 was labelled with ^(99m)Tcessentially as described by Samina Roohi et al., with minormodifications (Roohi S., et al., Journal of Radioanalytical and nuclearchemistry 267:561 (2006), (Roohi S., et al., Radiochim Acta 94:147(2006), (Roohi S., et al., Radiochim Acta 93:415 (2005)). Briefly,aliquots were thawed at room temperature and mixed with SnCl₂.2H₂O(Merck). 1 ml of Na^(99m)TcO₄ in saline (5-10 mCi/ml) was added in thevials and incubated for 10 min at room temperature. The effect oflabeling conditions i.e., pH, amount of reducing agents was alsodetermined. To determine the optimal amount of reducing agent, 5-100 μgof SnCl₂.2H₂O was used; pH was adjusted by using HCl/NaOH. The stabilityof ^(99m)Tc-CSA was checked for 4 hours at room temperature. Reactionmixture volume used in all studies was 1±0.1 ml. All the studies werecarried out at room temperature (22±2° C.).

Quality Control

Radiochemical yields of ^(99m)Tc-DTPA, ^(99m)Tc-Ciprofloxacin and^(99m)Tc-CSA were assessed by thin layer chromatographic methods. In thecase of ^(99m)Tc-DTPA free ^(99m)TcO₄ ⁻ was determined by using Whatmanpaper No. 3 as the stationary phase and acetone as the mobile phase.Reduced and hydrolyzed activity was determined by using Whatman paperno. 3 as the stationary phase and saline as mobile phase. Free^(99m)TcO₄ ⁻ in ^(99m)Tc-Ciprofloxacin was determined by using Whatmanpaper No. 3 as the stationary phase and acetone as the mobile phase.Reduced and hydrolyzed activity was determined by using Whatman paperno. 3 as the stationary phase and citrate buffer of pH 3.8 as mobilephase. In the case ^(99m)Tc-CSA free ^(99m)TcO₄ was determined by usingWhatman paper No. 3 as the stationary phase and acetone as the mobilephase. Reduced and hydrolyzed activity was determined by using instantthin layer chromatography (ITLC-SG strips) as the stationary phase and0.05 M NaOH as mobile phase. Radiocolloids were also determined bypassing the preparation through 0.22 μm sterile filters (MilliporeFilter Corp). Activity remaining on the filter and in solution wascounted in a gamma counter (Ludlum). The distribution of radioactivityon chromatographic stripes was measured by 2π Scanner (Berthold,Germany) or cut into 1 cm segments and counted in a gamma counter(Ludlum, USA).

Paper Electrophoresis

The charge of ^(99m)Tc-CSA was determined by paper electrophoresis usingNa-phosphate buffer of pH 6.8 as electrolyte and Whatman No. 1 assupport. The sample was run at a constant voltage of 300V for 1 h. Thestrip was cut into 1 cm pieces and counted in a well type gamma counter.For comparison, a sample of Na^(99m)TcO₄ was also run under identicalcondition.

Microorganism (Bacteria Staphylococcus aureus 25923)

S. aureus ATCC 25923 is the most widely used quality control organism inclinical microbiology laboratories. Being a standard reference strain,its use was similar to that of previous studies (Welling M. M., et al.,Eur J Nucl Med 27:292 (2000)). Overnight cultures of bacteria wereprepared in brain heart infusion broth (BHI, Oxoid) in a shaking waterbath at 37° C. Aliquots of suspensions containing viable stationaryphase bacteria were flash frozen in liquid nitrogen and stored at −70°C. Just before use, an aliquot of this suspension was rapidly thawed ina water bath at 37° C. and diluted in sodium phosphate buffer of pH 7.2(Na—PB).

In Vitro Binding of ^(99m)Tc-CSA to Bacteria

Binding of ^(99m)Tc-CSA to S. aureus bacteria was assessed by the methoddescribed elsewhere (Welling M. M., et al., Eur J Nucl Med 27:292(2000)). Briefly, 0.1 ml of sodium phosphate buffer (Na—PB) containing^(99m)Tc-CSA was transferred to a test tube. 0.8 ml of 50% (v/v) of 0.01M acetic acid in Na—PB containing approximately 1×10⁸ viable bacteriawas added. The mixture was incubated for 1 h at 4° C. and thencentrifuged for 5 min at 2000 rev/min. The supernatant was removed andthe bacterial pellet was gently resuspended in 1 ml of ice cooled Na—PBand recentrifuged. The supernatant was removed and the radioactivity inthe bacterial pellet was determined by a gamma-counter. The supernatantswere also counted. The radioactivity related to bacteria was expressedin percent of the added ^(99m)Tc activity bound to viable bacteria inregard to total ^(99m)Tc activity (Table 1). For comparison purposesbinding of ^(99m)Tc-DTPA and ^(99m)Tc-Ciprofloxacin to bacteria werealso performed following the reported procedures (Sonmezonglu K., etal., J Nucl Med 42:567 (2001); (Vinjamuri S., et al., Eur J Nucl Med347:233 (1996); (Oh S. J., et al., Appl Radiation Isotopes 57:193(2002)).

Thigh Muscle Infection

Male Sprague-Dawley rats weighing ˜200 g were used in all animalstudies. A turbid suspension containing 2×10⁸ colony-forming units (cfu)of S. aureus in 0.1 ml of saline was injected into the left thigh muscleof the rats. 48 h later, when visible swelling appeared in the infectedthigh, 0.2 ml of ^(99m)Tc-CSA (˜1 mCi) was injected via the tail vein.Four rats were used for one set of studies. Similar method was followedusing ^(99m)Tc-Ciprofloxacin.

Biodistribution Study

After a definite time, the rats were sacrificed after ether anesthesiaand biodistribution was determined. The whole animals were then weighedand dissected. Samples of infected muscle, normal muscle, liver, spleen,lung, kidney, stomach, and heart were weighed, and the activity wasmeasured using a gamma counter (Table 2). The results were expressed asthe percent uptake of injected dose per organ. The results of thebacterial uptake of ^(99m)Tc-CSA were analyzed by an analysis ofvariance and compared with ^(99m)Tc-Ciprofloxacin (Table 2). The levelof significance was set at 0.05.

^(99m)Tc-CSA Scintigraphy

Scintigraphy was performed in the rats infected with the S. aureusinfection in the thigh of left legs as described above. A single headed(Siemens Integrated ORBITER) Gamma Camera System interfaced withhigh-resolution parallel whole collimator was used. It was connected toan on-line dedicated computer (Macintosh® Operating System 7.5 Softwareused on the ICON™ Workstation). Each animal was placed on a flat hardsurface with both hind legs spread out and all legs fixed with surgicaltape. Saline (0.5 ml) containing ˜0.4 mCi of ^(99m)Tc-CSA was theninjected intravenously into the tail vein. Immediately after injection,dynamic acquisition with both thighs in focus was done for 60 min. Forthe biodistribution study of the radiotracer, whole body acquisition wasdone at 1 h and 4 h after injection.

Example 2

This example includes a description of radiolabeling of a CSA.

Labeling efficiency and radiochemical purity and stability were assessedby a combination of ascending paper chromatography and instant thinlayer chromatography on silica gel. In paper chromatography usingacetone as the solvent, free ^(99m)TcO₄ ⁻ moved towards the solventfront (Rf=1), while ^(99m)Tc-CSA and reduced/hydrolyzed ^(99m)Tcremained at the point of spotting. In ITLC-SG chromatography using 0.05MNaOH as the solvent, reduced/hydrolyzed ^(99m)Tc remained at the pointof spotting, whereas ^(99m)Tc-CSA and free ^(99m)TcO₄ ⁻ moved towardsthe solvent front. Radiocolloids were also determined by passing thepreparation through sterile filters (0.22 nm). In this techniqueradiocolloids were retained on the filter, while ^(99m)Tc-CSA and free^(99m)TcO₄ passed through. The results obtained by both methods were inexcellent agreement. The amount of radiocolloid in the finalpreparations was 2.0%.

The effects of pH are shown in FIG. 2. At low pH (2-4), the labelingefficiency was 100%, while at pH 7 the labeling efficiency of^(99m)Tc-CSA was <97%. In basic media at pH 10 the labeling efficiencywas decreased to 95%. Hence further studies were performed at pH 4.

The amount of the reducing agent, SnCl₂.2H₂O, which gave the highestlabeling efficiency, was 15-60 μg (FIG. 3). To avoid colloid formation,the optimum amount of reducing agent (30 μg) was used. The complexationof ^(99m)Tc with CSA was rapid and maximum labeling efficiency wasachieved after 10 min. The resulting complex of ^(99m)Tc-CSA was quitestable and labeling of ≥98% is maintained for up to 6 h (FIG. 4). Paperelectrophoresis showed that the ^(99m)Tc-CSA species does not move tocathode or anode indicating that the compound exhibits neutral behavior(FIG. 5). The final formulation for the radiotracer ^(99m)Tc-CSA was:CSA: 200 μg; SnCl₂.2H₂O: 30 μg; pH 4; ^(99m)Tc ˜10 mCi; reaction mixturevolume ˜1 ml; and incubation time 10 min at room temperature.Radiolabeling efficiency of ^(99m)Tc-CSA monitored by paper and ITLC-SGwas higher than 97%. The resulting complex of ^(99m)Tc-CSA is quitestable and labeling of ≥98% is maintained for up to 4 h. Nopost-labeling purification was required. The biological activity (invitro) of ^(99m)Tc-CSA is significantly higher than^(99m)Tc-Ciprofloxacin. ^(99m)Tc-CSA is able to localize in bacterialinfection induced by S. aureus in animal models and may be used in avariety of patients referred for infection

Example 3

This Example describes in vitro and in vivo binding of labeled CSA toStaphylococcus aureus.

In vitro binding of 10 μg of ^(99m)Tc-CSA was in the range of 96.3% at0.5 h, which decreased to 90% after 24 h. In-vitro binding of 50 μg and100 μg ^(99m)Tc-CSA was 70% and 63% respectively at 30 min; however,maximum binding with S. aureus was achieved after 24 h (>90%) (FIG. 6).In-vitro binding of ^(99m)Tc-Ciprofloxacin, a promising agent for thediagnosis of bacterial infection, to bacteria at 50 μg^(99m)Tc-Ciprofloxacin concentration was observed as 70%±4.0 (FIG. 7,Table 1). In vitro binding of ^(99m)Tc-DTPA (kidney/brain imaging agent)to bacteria at 10 μg ^(99m)Tc-DTPA concentration was ˜10% (FIG. 7, Table1). In published reportes, the in-vitro binding of^(99m)Tc-Ciprofloxacin and ^(99m)Tc-DTPA ranged from 40 to 65% and >8%respectively (Obradovic, V., et al., World J Nuc. Med 2:269 (2003),(Corn, M. J., et al., Acad Sci 76:27 (1958)).

TABLE 1 Table 1. In-vitro binding of ^(99m)Tc-CSA to viable S. aureus incomparison with ^(99m)Tc-Ciprofloxacin (n = 4 per study). Conc. Time(hrs) (μg) 0.5 1 4 24 ^(99m)Tc-CSA 10 96.3 ± 3.0  99.4 ± 0.05  90 ± 5.090.6 ± 6.67  50 70 ± 6.6  73 ± 2.78  83 ± 4.75 92 ± 7.70 100  63 ± 5.0560 ± 6.0 72 ± 5.9 94 ± 4.05 ^(99m)Tc- 10 Ciprofloxacin 50 70 ± 4.0 73 ±4.0 75 ± 4.5 73 ± 5.0  100

The tissue distribution of ^(99m)Tc-CSA in rats with bacterial infectionwas studied at 1 and 4 h after intravenous administration, and resultsare presented in Table 2. The tissue distribution is expressed aspercentage of injected dose per organ (% ID/organ). The ^(99m)Tc-CSA wasrapidly distributed after intravenous injection and liver and spleenuptake was significant after 1 h and remained high after 4 h. However,the lung lung shows a significant uptake after 4 h. It is assumed that^(99m)Tc-CSA is stable in vivo, since insignificant activity was noticedin thyroid and stomach during biodistribution studies. FIG. 8 shows theinfected thigh muscles and normal thigh muscles obtained at 4 h afteradministration of ^(99m)Tc-CSA. The infected thigh/normal thighradioactivity ratio indicated that higher binding affinity to theinfection induced with S. aureus was observed. The highesttarget/non-target ratio reached 1.6 at 1 h and remained 2.4 at 4 h postinjection of ^(99m)Tc-CSA, comparable to the ^(99m)Tc-Ciprofloxacinwhich showed a value of 2.0 at 4 hours post injection time (Table 2).

TABLE 2 Table 2. Biodistribution data in percent injected dose per organfor ^(99m)Tc-CSA and ^(99m)Tc-Ciprofloxacin after 4 hours post injectionin infected Sprauge-Dawley rats (n = 4). Organ ^(99m)Tc-CSA^(99m)Tc-Ciprofloxacin Infected thigh/ 2.3 ± 0.3 2.0 ± 0.5 Normal thighLiver  93 ± 0.8 13.4 ± 0.8  Spleen 1.22 ± 0.05 0.4 ± 0.0 Stomach 0.080 ±0.02  0.6 ± 0.1 Lungs 0.41 ± 0.09 0.3 ± 0.0 Kidney 0.52 ± 0.05 8.9 ± 0.4Bladder 0.016 ± 0.0  42.98 ± 4.0  Heart 0.066 ± 0.01  0.1 ± 0.0

A whole body image of the infected rat at 4 hours after ^(99m)Tc-CSAadministration is presented in FIG. 8. S. aureus infection in rat thighwas visualized as area of increased tracer accumulation after injectionof labeled CSA. The infection was not clearly visible at 1 h postadministration, whereas it was clearly visible after 4 hours.Target-to-background ratios obtained from region of interest analysis of^(99m)Tc-CSA ranged 1.6 to 2.4. In vitro studies and animal studies haveshown that ^(99m)Tc-CSA localizes in bacteria infected sitessignificantly. Due to the ease of ^(99m)Tc-CSA preparation and infectionuptake, it may be used similar to ^(99m)Tc-Ciprofloxacin in a variety ofpatients referred for infection (Britton K. E., et al., Eur J Nucl Med24:553 (1997); (Das, S. S., et al., World J Nucl Med 2:173 (2003);(Larikka, M. J., et al., Nucl Med Commun 23:167 (2002); (Corstens, F.H., et al., Lancet 354 (1997)). However; further studies are needed inthis direction.

Example 4

This example includes a description of one or more exemplary synthesticprocedures for obtaining Compounds 1-5, 13-20 and 22-27.

Compound 13: To a 1 L round-bottom flask were added methyl cholate(30.67 g, 72.7 mmol) in dry THF (600 mL) and LiAlH₄ (4.13 g, 109 mmol).After reflux for 48 hours, saturated aqueous Na₂SO₄ (100 mL) wasintroduced slowly, and the resulted precipitate was filtered out andwashed with hot THF and MeOH. Recrystallization from MeOH gave colorlesscrystals of 13 (28.0 g, 98% yield). m.p. 236.5-238° C.; IR (KBr) 3375,2934, 1373, 1081 cm⁻¹; ¹H NMR (CDCl₃/MeOH-d₄, 200 MHz) δ 3.98 (bs, 1H),3.83 (bs, 1H), 3.60-3.46 (m, 2H), 3.38 (bs, 5H), 2.30-2.10 (m, 2H),2.05-1.05 (series of multiplets, 22H), 1.03 (bs, 3H), 0.92 (s, 3H), 0.71(s, 3H); ¹³C NMR (CDCl₃/MeOH-d₄, 50 MHz) δ 73.89, 72.44, 68.99, 63.51,48.05, 47.12, 42.49, 40.37, 39.99, 36.62, 36.12, 35.58, 35.40, 32.77,30.69, 30.04, 29.02, 28.43, 27.27, 23.96, 23.08, 18.00, 13.02; HRFAB-MS(thioglycerol+Na⁺ matrix) m/e: ([M+Na]⁺) 417.2992 (55.3%); calcd.417.2981.

Compound 14: To a round-bottom flask were added 13 (28.2 g, 71.7 mmol)in DMF (300 ml), Et₃N (20 mL, 143.4 mmol), trityl chloride (25.98 g,93.2 mmol) and DMAP (0.13 g, 1.07 mmol). The mixture was stirred at 50°C. under N₂ for 30 hours followed by the introduction of water (1000 mL)and extraction with EtOAc (5×200 mL). The combined extracts were washedwith water and brine and then dried over MgSO₄. After removal of solventin vacuo, the residue was purified using SiO₂ chromatography (CH₂Cl₂,Et₂O and MeOH as eluents) to give 14 as a pale yellow solid (31.9 g, 70%yield). m.p. 187° C. (decomposition); IR (KBr) 3405, 2935, 1448, 1075cm⁻¹; ¹H NMR (CDCl₃, 200 MHz) δ 7.46-7.42 (m, 6H), 7.32-7.17 (m, 9H),3.97 (bs, 1H), 3.83 (bs, 1H), 3.50-3.38 (m, 1H), 3.01 (bs, 1H), 2.94(dd, J=14.2, 12.2 Hz, 2H), 2.64 (bs, 1H), 2.51 (bs, 1H), 2.36-2.10 (m,2H), 2.00-1.05 (series of multiplets, 22H), 0.96 (d, J=5.8 Hz, 3H), 0.87(s, 3H), 0.64 (s, 3H); ¹³C NMR (CDCl₃, 50 MHz) δ 144.77, 128.93, 127.91,127.01, 86.43, 73.35, 72.06, 68.66, 64.28, 47.47, 46.53, 41.74, 41.62,39.64, 35.57, 35.46, 34.91, 34.82, 32.40, 30.55, 28.21, 27.69, 26.80,26.45, 23.36, 22.59, 17.83, 12.61; HRFAB-MS (thioglycerol+Na⁺ matrix)m/e: ([M+Na]⁺) 659.4069 (100%); calcd. 659.4076.

Compound 15: To a round-bottom flask were added 14 (20.0 g, 31.4 mmol)in dry THF (600 mL) and NaH (60% in mineral oil, 6.3 g, 157.2 mmol). Themixture was refluxed for 30 min under N₂ followed by addition of allylbromide (27 mL, 314 mmol). After 60 hours of reflux, additional NaH (3eq.) and allyl bromide (4 eq.) were added. Following another 50 hours ofreflux, water (20 mL) was introduced slowly followed by addition of 1%HCl until the aqueous layer became neutral. The mixture was thenextracted with ether (3×100 mL) and the combined extracts were washedwith water (100 mL) and brine (2×100 mL). The ether solution was driedover anhydrous Na₂SO₄, and after removal of solvent, the residue waspurified using SiO₂ chromatography (hexanes and EtOAc/hexanes 1:8 aseluents) to give 15 (22.76 g, 96% yield) as a pale yellow glass. IR(neat) 2930, 1448, 1087 cm⁻¹; ¹H NMR (CDCl₃, 200 MHz) δ 7.48-7.30 (m,6H), 7.32-7.14 (m, 9H), 6.04-5.80 (m, 3H), 5.36-5.04 (series ofmultiplets, 6H), 4.14-3.94 (m, 4H), 3.74 (td, J=13.8, 5.8 Hz, 2H), 3.53(bs, 1H), 3.20-2.94 (m, 3H), 3.31 (bs, 1H), 2.38-1.90 (m, 4H), 1.90-0.96(series of multiplets, 20H), 0.90 (d, J=5.4 Hz, 3H), 0.89 (s, 3H), 0.64(s, 3H); ¹³C NMR (CDCl₃, 50 MHz) δ 144.83, 136.27, 136.08, 128.94,127.90, 126.98, 116.46, 115.70, 86.42, 80.94, 79.29, 74.98, 69.52,69.39, 68.86, 64.39, 46.51, 46.42, 42.67, 42.14, 39.92, 35.63, 35.51,35.13, 32.45, 28.98, 28.09, 27.66, 27.57, 26.72, 23.32, 23.11, 17.92,12.69; HRFAB-MS (thioglycerol+Na⁺ matrix) m/e: ([M+Na]⁺) 779.5013(86.1%); calcd. 779.5015.

Compound 16: To a three-necked round bottom flask was added 15 (3.34 g,4.4 mmol) in CH₂Cl₂ (200 mL) and methanol (100 mL). Through the coldsolution (˜78° C.) ozone was bubbled through until a blue colorpersisted. Excess ozone was removed with oxygen flow. The mixture wasleft in a dry ice-acetone bath for an hour. Methyl sulfide (2.4 mL) wasadded and 15 minutes later, the mixture was treated with NaBH₄ (1.21 g,32 mmol) in 5% aqueous NaOH solution (10 mL)/methanol (10 mL) andallowed to warm to room temperature. The mixture was washed with brine(3×50 mL), and the combined brine wash was extracted with CH₂Cl₂ (2×50mL). The organic solution was dried over MgSO₄. After SiO₂chromatography (MeOH (5%) in CH₂Cl₂), 3.30 g (95% yield) of 16 wasisolated as an oil. IR (neat) 3358, 2934, 1448, 1070 cm⁻¹; ¹H NMR(CDCl₃, 200 MHz) δ 7.50-7.42 (m, 6H), 7.32-7.17 (m, 9H), 3.80-2.96(series of multiplets, 20H), 2.25-0.96 (series of multiplets, 24H), 0.89(bs, 6H), 0.65 (s, 3H); ¹³C NMR (CDCl₃, 50 MHz) δ 144.73, 128.88,127.87, 126.96, 86.38, 81.05, 79.75, 76.59, 70.33, 69.66, 69.30, 64.20,62.25, 62.16, 62.03, 46.77, 46.36, 42.63, 41.77, 39.60, 35.43, 35.23,35.05, 34.89, 32.42, 28.91, 27.93, 27.56, 27.15, 26.68, 23.35, 22.98,22.85, 18.15, 12.60; HRFAB-MS (thioglycerol+Na⁺ matrix) m/e: ([M+Na]⁺)791.4860 (100%), calcd. 791.4863.

Compound 17: To a round-bottom flask was added 16 (1.17 g, 1.55 mmol) indry THF (30 mL) under N₂ in ice-bath followed by 9-BBN/THF solution (0.5M, 10.2 mL, 5.51 mmol). The mixture was stirred at room temperature for12 hours. Aqueous NaOH (20%) (2 mL) and hydrogen peroxide (30%) (2 mL)were added in sequence. The mixture was refluxed for 1 hour followed bythe addition of brine (60 mL) and extraction with EtOAc (4×30 mL). Thecombined extracts were dried over anhydrous Na₂SO₄. The product (1.01 g,80% yield) was obtained as a colorless oil after SiO₂ chromatography (5%MeOH in CH₂Cl₂). IR (neat) 3396, 2936, 1448, 1365, 1089 cm⁻¹; ¹H NMR(CDCl₃, 200 MHz) δ 7.50-7.42 (m, 6H), 7.34-7.16 (m, 9H), 3.90-3.56 (m,13H), 3.50 (bs, 1H), 3.40-2.96 (series of multiplets, 6H), 2.30-0.94(series of multiplets, 30H), 0.90 (s, 3H), 0.88 (d, J=5.4 Hz, 3H), 0.64(s, 3H); ¹³C NMR (CDCl₃, 50 MHz) δ 144.73, 128.88, 127.85, 126.94,86.36, 80.52, 78.90, 76.36, 66.82, 66.18, 65.77, 64.22, 61.53, 61.41,61.34, 46.89, 46.04, 42.60, 41.59, 39.60, 35.37, 35.27, 34.88, 32.75,32.44, 32.31, 28.82, 27.65, 27.48, 27.13, 26.77, 23.35, 22.74, 22.38,18.08, 12.48; HRFAB-MS (thioglycerol+Na⁺ matrix) m/e: ([M+Na]⁺) 833.5331(100%), calcd. 833.5332.

Compound 18: To a round-bottom flask were added 16 (3.30 g, 4.29 mmol)in CH₂Cl₂ (150 mL) and NEt₃ (2.09 mL, 15.01 mmol). The mixture was putin ice-bath under N₂ followed by addition of mesyl chloride (1.10 mL,14.16 mmol). After 30 minutes, water (30 mL) and brine (200 mL) wereadded. The CH₂Cl₂ layer was washed with brine (2×50 mL) and dried overanhydrous Na₂SO₄. The combined aqueous mixture was extracted with EtOAc(3×100 mL). The combined extracts were washed with brine and dried overanhydrous Na₂SO₄. The desired product (3.35 g, 78% yield) was isolatedas a pale yellow oil after SiO₂ chromatography (EtOAc/hexanes 1:1). IR(neat) 2937, 1448, 1352, 1174, 1120, 924 cm⁻¹; ¹H NMR (CDCl₃, 200 MHz) δ7.52-7.40 (m, 6H), 7.34-7.20, (m, 9H), 4.42-4.24 (m, 6H), 3.90-3.64 (m,4H), 3.60-3.30 (m, 4H), 3.24-3.00 (m, 3H), 3.10 (s, 6H), 3.05 (s, 3H),2.20-1.96 (m, 3H) 1.96-1.60 (m, 8H), 1.60-0.94 (series of multiplets,13H), 0.91 (bs, 6H), 0.65 (s, 3H); ¹³C NMR (CDCl₃, 50 MHz) δ 114.68,128.85, 127.85, 126.96, 86.37, 81.37, 79.58, 76.58, 69.95, 69.43, 69.34,66.52, 66.31, 65.59, 64.11, 46.80, 46.20, 42.65, 41.48, 39.35, 37.82,37.48, 35.36, 34.92, 34.73, 32.37, 28.66, 28.01, 27.44, 27.03, 26.72,23.17, 22.91, 22.72, 18.13, 12.50; HRFAB-MS (thioglycerol+Na⁺ matrix)m/e: ([M+Na]⁺) 1205.4176 (81.5%), calcd. 1205.4189.

Compound 19: To a round-bottom flask were added 17 (1.01 g, 1.25 mmol)in CH₂Cl₂ (50 mL) and NEt₃ (0.608 mL, 4.36 mmol). The mixture was put inice-bath under N₂ followed by addition of mesyl chloride (0.318 mL, 4.11mmol). After 30 minutes, water (10 mL) and then brine (80 mL) wereadded. The CH₂Cl₂ layer was washed with brine (2×20 mL) and dried overanhydrous Na₂SO₄. The combined aqueous mixture was extracted with EtOAc(3×40 mL). The combined extracts were washed with brine and dried overanhydrous Na₂SO₄. The desired product (1.07 g, 82%) was isolated as apale yellowish oil after SiO₂ chromatography (EtOAc/hexanes 1:1). IR(neat) 2938, 1356, 1176, 1112 cm⁻¹; ¹H NMR (CDCl₃, 300 MHz) δ 7.46-7.43,(m, 6H), 7.32-7.22 (m, 9H), 4.40-4.31 (m, 6H), 3.72-3.64 (m, 2H), 3.55(dd, J=6.3, 5.8 Hz, 2H), 3.51 (bs, 1H), 3.32-3.14 (m, 3H), 3.14-2.92 (m,3H), 3.01 (s, 3H), 3.01 (s, 3H), 3.00 (s, 3H), 2.10-1.92 (m, 10H),1.92-1.58 (m, 8H), 1.56-0.92 (series of multiplets, 12H), 0.90 (s, 3H),0.89 (d, J=5.4 Hz, 3H), 0.64 (s, 3H); ¹³C NMR (CDCl₃, 75 MHz) δ 144.67,128.85, 127.85, 126.96, 86.42, 81.06, 79.83, 76.81, 68.12, 68.06, 68.02,64.26, 64.06, 63.42, 46.76, 46.38, 42.73, 41.87, 39.73, 37.44, 37.32,37.29, 35.52, 35.48, 35.32, 35.06, 32.53, 30.55, 30.28, 30.02, 29.15,27.96, 27.69, 27.61, 26.75, 23.52, 23.02, 18.17, 12.64; HRFAB-MS(thioglycerol+Na⁺ matrix) m/e: ([M+Na]⁺) 1067.4672 (100%), calcd.1067.4659.

Compound 20: To a round-bottom flask were added 18 (1.50 g, 1.50 mmol)in dry DMSO (20 mL) and NaN₃ (0.976 g, 15 mmol). The mixture was heatedto 80° C. and stirred under N₂ overnight then diluted with water (100mL). The resulted aqueous mixture was extracted with EtOAc (3×50 mL),and the combined extracts washed with brine and dried over anhydrousNa₂SO₄. The desired product (0.83 g, 66% yield) was isolated as a clearglass after SiO₂ chromatography (EtOAc/hexanes 1:5). IR (neat) 2935,2106, 1448, 1302, 1114 cm⁻¹; ¹H NMR (CDCl₃, 200 MHz) δ 7.50-7.42 (m,6H), 7.36-7.20 (m, 9H), 3.84-3.70 (m, 2H), 3.65 (t, J=4.9 Hz, 2H), 3.55(bs, 1H), 3.44-3.08 (m, 10H), 3.02 (t, J=6.4 Hz, 2H), 2.38-0.96 (seriesof multiplets, 24H), 0.92 (d, J=5.6 Hz, 3H), 0.91 (s, 3H), 0.65 (s, 3H);¹³C NMR (CDCl₃, 50 MHz) δ 114.84, 128.97, 127.92, 126.99, 86.42, 81.24,80.12, 76.59, 67.84, 67.29, 66.66, 64.36, 51.67, 51.44, 51.18, 46.53,46.23, 42.21, 41.93, 39.73, 35.66, 35.36, 35.06, 34.78, 32.40, 28.95,27.76, 27.39, 26.87, 23.45, 22.98, 22.92, 17.98, 12.53; HRFAB-MS(thioglycerolNa⁺ matrix) m/e: ([M+Na]⁺) 866.5040 (100%), calcd.866.5057.

Compound 22: To a round-bottom flask were added 20 (830 mg, 0.984 mmol)in MeOH (30 mL) and CH₂Cl₂ (30 mL) and p-toluenesulfonic acid (9.35 mg,0.0492 mmol). The solution was stirred at room temperature for 2.5 hoursthen saturated aqueous NaHCO₃ (10 mL) was introduced. Brine (30 mL) wasadded, and the mixture was extracted with EtOAc (4×20 mL). The combinedextracts were dried over anhydrous Na₂SO₄. The desired product (0.564 g,95% yield) was isolated as a pale yellowish oil after SiO₂chromatography (EtOAc/hexanes 1:2). IR (neat) 3410, 2934, 2106, 1301,1112 cm⁻¹; ¹H NMR (CDCl₃, 200 MHz) δ 3.80-3.54 (m, 7H), 3.44-3.20 (m,10H), 2.35-0.96 (series of multiplets, 24H), 0.95 (d, J=6.4 Hz, 3H),0.92 (s, 3H), 0.68 (s, 3H); ¹³C NMR (CDCl₃, 50 MHz) δ 81.10, 80.01,76.60, 67.75, 67.16, 66.56, 63.63, 51.57, 51.34, 51.06, 46.29, 46.12,42.12, 41.81, 39.60, 35.55, 35.23, 34.94, 34.66, 31.75, 29.48, 28.81,27.72, 27.66, 27.29, 23.32, 22.86, 22.80, 17.85, 12.39; HRFAB-MS(thioglycerol+Na⁺ matrix) m/e: ([M+Na]⁺) 624.3965 (100%), calcd.624.3962.

Compound 23: To a round-bottom flask were added 19 (1.07 g, 1.025 mmol)and NaN₃ (0.666 g, 10.25 mmol) followed the introduction of dry DMSO (15mL). The mixture was heated up to 80° C. under N₂ overnight. After theaddition of H₂O (100 mL), the mixture was extracted with EtOAc (4×40 mL)and the combined extracts were washed with brine (2×50 mL) and driedover anhydrous Na₂SO₄. After removal of solvent, the residue wasdissolved in MeOH (15 mL) and CH₂Cl₂ (15 mL) followed by the addition ofcatalytic amount of p-toluenesulfonic acid (9.75 mg, 0.051 mmol). Thesolution was stirred at room temperature for 2.5 hours before theaddition of saturated NaHCO₃ solution (15 mL). After the addition ofbrine (60 mL), the mixture was extracted with EtOAc (5×30 mL). Thecombined extracts were washed with brine (50 mL) and dried overanhydrous Na₂SO₄. The desired product (0.617 g, 94% yield for two steps)was obtained as a yellowish oil after SiO₂ chromatography (EtOAc/hexanes1:2). IR (neat) 3426, 2928, 2094, 1456, 1263, 1107 cm⁻¹; ¹H NMR (CDCl₃,300 MHz) δ 3.68-3.56 (m, 3H), 3.56-3.34 (series of multiplets, 10H),3.28-3.00 (series of multiplets, 4H), 2.20-2.00 (m, 3H), 1.98-1.55(series of multiplets, 15H), 1.55-0.96 (series of multiplets, 13H), 0.92(d, J=6.6 Hz, 3H), 0.89 (s, 3H), 0.66 (s, 3H); ¹³C NMR (CDCl₃, 75 MHz) δ80.63, 79.79, 76.04, 64.99, 64.45, 64.30, 63.72, 49.01, 48.94, 48.74,46.49, 46.39, 42.70, 41.98, 39.80, 35.65, 35.42, 35.28, 35.08, 31.99,29.78, 29.75, 29.70, 29.49, 29.06, 27.87, 27.79, 27.65, 23.53, 23.04,22.85, 18.05, 12.59; HRFAB-MS (thioglycerol+Na matrix) m/e: ([M+Na]⁺)666.4415 (100%), calcd. 666.4431.

Compound 24: To a round-bottom flask were added 22 (0.564 g, 0.938 mmol)in CH₂Cl₂ (30 mL) and NEt₃ (0.20 mL, 1.40 mmol). The mixture was put inice-bath under N₂ followed by addition of mesyl chloride (0.087 mL, 1.13mmol). After 30 minutes, water (20 mL) and brine (100 mL) were added.The CH₂Cl₂ layer was washed with brine (2×20 mL) and dried overanhydrous Na₂SO₄. The combined aqueous mixture was extracted with EtOAc(3×30 mL). The combined extracts were washed with brine and dried overanhydrous Na₂SO₄. The desired product (0.634 g, 99% yield) was isolatedas a pale yellowish oil after SiO₂ chromatography (EtOAc/hexanes 1:2).IR (neat) 2935, 2106, 1356, 1175, 1113 cm⁻¹; ¹H NMR (CDCl₃, 300 MHz) δ4.20 (t, J=6.8 Hz, 2H), 3.80-3.75 (m, 1H), 3.70-3.64 (m, 3H), 3.55 (bs,1H), 3.44-3.01 (m, 10H), 3.00 (s, 3H), 2.32-2.17 (m, 3H), 2.06-2.03 (m,1H), 1.90-0.88 (series of multiplets, 20H), 0.95 (d, J=6.6 Hz, 3H), 0.91(s, 3H), 0.68 (s, 3H); ¹³C NMR (CDCl₃, 75 MHz) δ 80.90, 79.86, 76.43,70.78, 67.64, 66.99, 66.48, 51.50, 51.26, 50.97, 46.05, 45.96, 42.08,41.71, 39.51, 37.33, 35.15, 34.86, 34.60, 31.34, 28.73, 27.62, 27.59,27.51, 25.68, 23.22, 22.80, 22.70, 17.62, 12.33; HRFAB-MS(thioglycerol+Na⁺ matrix) m/e: ([M+Na]⁺) 702.3741 (100%), calcd.702.3737.

Compound 25: To a round-bottom flask were added 23 (0.617 g, 0.96 mmol)in CH₂Cl₂ (30 mL) and NEt₃ (0.20 mL, 1.44 mmol). The mixture was put inice-bath under N₂ followed by addition of mesyl chloride (0.089 mL, 1.15mmol). After 30 minutes, water (20 mL) and brine (120 mL) were added.The CH₂Cl₂ layer was washed with brine (2×20 mL) and dried overanhydrous Na₂SO₄. The combined aqueous mixture was extracted with EtOAc(3×30 mL). The combined extracts were washed with brine and dried overanhydrous Na₂SO₄. The desired product (0.676 g, 97% yield) was isolatedas a pale yellowish oil after removal of solvent. IR (neat) 2934, 2094,1454, 1360, 1174, 1112 cm⁻¹; ¹H NMR (CDCl₃, 300 MHz) δ 4.17 (t, J=6.6Hz, 2H), 3.65-3.28 (series of multiplets, 11H), 3.64-3.00 (series ofmultiplets, 4H), 2.97 (s, 3H), 2.18-1.96 (series of multiplets, 16H),1.54-0.94 (series of multiplets, 11H), 0.89 (d, J=6.6 Hz, 3H), 0.86 (s,3H), 0.63 (s, 3H); ¹³C NMR (CDCl₃, 75 MHz) δ 80.47, 79.67, 75.92, 70.84,64.90, 64.37, 64.17, 48.90, 48.86, 48.66, 46.32, 46.26, 42.63, 41.87,39.70, 37.39, 35.34, 35.28, 35.20, 34.99, 31.61, 29.68, 29.60, 28.96,27.78, 27.68, 27.57, 25.79, 23.41, 22.95, 22.74, 17.82, 12.50; HRFAB-MS(thioglycerol matrix) m/e: ([M+H]⁺) 722.4385 (22.1%), calcd. 722.4387.

Compound 26: To a 50 mL round-bottom flask was added 24 (0.634 g, 0.936mmol) and N-benzylmethylamine (2 mL). The mixture was heated under N₂ at80° C. overnight. Excess N-benzylmethylamine was removed under vacuum,and the residue was subjected to SiO₂ chromatography (EtOAc/hexanes1:2). The desired product (0.6236 g, 95% yield) was isolated as a paleyellow oil. IR (neat) 2935, 2106, 1452, 1302, 1116 cm⁻¹; ¹H NMR (CDCl₃,200 MHz) δ 7.32-7.24 (m, 5H), 3.80-3.76 (m, 1H), 3.70-3.60 (m, 3H), 3.54(bs, 1H), 3.47 (s, 2H), 3.42-3.10 (m, 10H), 2.38-2.05 (m, 5H), 2.17 (s,3H), 2.02-0.88 (series of multiplet, 21H), 0.93 (d, J=7.0 Hz, 3H), 0.91(s, 3H), 0.66 (s, 3H); ¹³C NMR (CDCl₃, 50 MHz) δ 139.60, 129.34, 128.38,127.02, 81.22, 80.10, 76.71, 67.85, 67.29, 66.65, 62.45, 58.38, 51.65,51.44, 51.16, 46.50, 46.21, 42.40, 42.20, 41.93, 39.72, 35.80, 35.34,35.05, 34.76, 33.65, 28.93, 27082, 27.75, 27.38, 24.10, 23.45, 22.98,22.91, 18.05, 12.50; HRFAB-MS (thioglycerol+Na⁺ matrix) m/e: ([M−H]⁻)703.4748 (90.2%), calcd. 703.4772; ([M+H]⁺) 705.4911 (100%), calcd.705.4928; ([M+Na]⁺) 727.4767 (1.5%), calcd. 727.4748.

Compound 27: To a 50 mL round-bottom flask was added 25 (0.676 g, 0.937mmol) and N-benzylmethylamine (2 mL). The mixture was heated under N₂ at80° C. overnight. Excess N-benzylmethylamine was removed under vacuumand the residue was subjected to SiO₂ chromatography (EtOAc/hexanes1:2). The desired product (0.672 g, 96% yield) was isolated as a paleyellow oil. IR (neat) 2934, 2096, 1452, 1283, 1107 cm⁻¹; ¹H NMR (CDCl₃,300 MHz) δ 7.34-7.20 (m, 5H), 3.68-3.37 (series of multiplets, 13H),3.28-3.04 (m, 4H), 2.33 (t, J=7.0 Hz, 2H), 2.18 (s, 3H), 2.20-2.00 (m,3H), 1.96-1.56 (series of multiplets, 14H), 1.54-1.12 (m, 10H),1.10-0.96 (m, 3H), 0.91 (d, J=8.7 Hz, 3H), 0.89 (s, 3H), 0.65 (s, 3H);¹³C NMR (CDCl₃, 75 MHz) δ 139.48, 129.23, 128.30, 126.96, 80.66, 79.81,76.08, 65.00, 64.46, 64.34, 62.50, 58.37, 49.02, 48.95, 48.75, 46.65,46.40, 42.69, 42.43, 42.00, 39.83, 35.86, 35.45, 35.30, 35.10, 33.83,29.81, 29.78, 29.72, 29.09, 27.88, 27.81, 27.66, 24.19, 23.57, 23.06,22.87, 18.15, 12.62; HRFAB-MS (thioglycerol matrix) m/e: ([M+H]⁺)747.5406 (77.2%), calcd. 747.5398.

Compound 1: To a round-bottom flask were added 26 (0.684 g, 0.971 mmol)in dry THF (30 mL) and LiAlH₄ (113.7 mg, 3.0 mmol) under N₂. The mixturewas stirred at room temperature for 12 hours, and then Na₂SO₄O.10 H₂Opowder (10 g) was added slowly. After the grey color disappeared, themixture was filtered through Celite and washed with dry THF. The product(0.581 g, 95% yield) was obtained as a colorless glass. IR (neat) 3372,2937, 1558, 1455, 1362, 1102 cm⁻¹; H NMR (CDCl₃, 300 MHz) δ 7.34-7.20(m, 5H), 3.68-3.48 (m, 5H), 3.47 (s, 2H), 3.29 (bs, 1H), 3.22-3.00 (m,3H), 2.96-2.80 (m, 6H), 2.32 (t, J=6.8, 5.4 Hz, 2H), 2.17 (s, 3H),2.20-2.00 (m, 3H), 1.96-0.96 (series of multiplets, 27H), 0.93 (d, J=6.8Hz, 3H), 0.90, (s, 3H), 0.67 (s, 3H); ¹³C NMR (CDCl₃, 75 MHz) δ 139.50,129.22, 128.31, 126.96, 80.76, 79.85, 76.10, 70.90, 70.33, 70.24, 62.48,58.27, 46.55, 46.45, 42.72, 42.58, 42.33, 41.99, 39.77, 35.78, 35.37,35.01, 33.73, 29.07, 27.95, 27.71, 24.06, 23.46, 22.99, 18.14, 12.55;HRFAB-MS (thioglycerol matrix) m/e: ([M+H]⁺) 627.5211 (100%), calcd.627.5213.

HCl salt of compound 1: Compound 1 was dissolved in a minimum amount ofCH₂Cl₂ and excess HCl in ether was added. Solvent and excess HCl wereremoved in vacuo and a noncrystalline white powder was obtained. ¹H NMR(methanol-d4/15% (CDCl₃, 300 MHz) δ 7.61-7.57 (m, 2H), 7.50-7.48 (m,3H), 4.84 (bs, 10H), 4.45 (bs, 1H), 4.30 (bs, 1H), 3.96-3.82 (m, 2H),3.78-3.69 (m, 2H), 3.66 (bs, 1H), 3.59-3.32 (series of multiplets, 4H),3.28-3.02 (m, 8H), 2.81 (s, 3H), 2.36-2.15 (m, 4H), 2.02-1.68 (m, 8H),1.64-0.90 (series of multiplets, 12H), 1.01 (d, J=6.35 Hz, 3H), 0.96 (s,3H), 0.73 (s, 3H); ¹³C NMR (methanol-d4/15% (CDCl₃, 75 MHz) δ 132.31,131.20, 130.92, 130.40, 83.13, 81.09, 78.48, 65.54, 64.98, 64.11, 60.87,57.66, 47.51, 46.91, 43.52, 43.00, 41.38, 41.19, 41.16, 40.75, 40.30,36.37, 36.08, 36.00, 35.96, 33.77, 29.68, 29.34, 28.65, 28.37, 24.42,24.25, 23.33, 21.51, 18.80, 13.04.

Compound 2: To a round-bottom flask were added 27 (0.82 g, 1.10 mmol) indry THF (150 mL) and LiAlH₄ (125 mg, 3.30 mmol) under N₂. The mixturewas stirred at room temperature for 12 hours and Na₂SO₄.10 H₂O powder(10 g) was added slowly. After the grey color disappeared, the mixturewas filtered through a cotton plug and washed with dry THF. THF wasremoved in vacuo and the residue dissolved in CH₂Cl₂ (50 mL). Afterfiltration, the desired product was obtained as a colorless glass (0.73g, 99% yield). IR (neat) 3362, 2936, 2862, 2786, 1576, 1466, 1363, 1103cm⁻¹; ¹H NMR (CDCl₃, 300 MHz) δ 7.32-7.23 (m, 5H), 3.67-3.63 (m, 1H),3.60-3.57 (m, 1H), 3.53 (t, J=6.4 Hz, 2H), 3.47 (s, 2H), 3.46 (bs, 1H),3.24-3.17 (m, 2H), 3.12-2.99 (m, 2H), 2.83-2.74 (series of multiplets,6H), 2.30 (t, J=7.3 Hz, 2H), 2.15 (s, 3H), 2.20-2.00 (m, 3H), 1.95-1.51(series of multiplets, 20H), 1.51-1.08, (series of multiplets, 10H),1.06-0.80 (m, 3H), 0.87 (d, J=8.1 Hz, 3H), 0.86 (s, 3H), 0.61 (s, 3H);¹³C NMR (CDCl₃, 75 MHz). 139.35, 129.16, 128.22, 126.88, 80.44, 79.29,75.96, 66.70, 66.52, 66.12, 62.45, 58.26, 46.76, 46.27, 42.69, 42.41,42.02, 40.68, 40.10, 40.02, 39.82, 35.84, 35.47, 35.30, 35.06, 34.15,34.09, 34.03, 33.80, 28.96, 27.93, 27.75, 27.71, 24.32, 23.53, 23.03,22.75, 18.17, 12.58; HRFAB-MS (thioglycerol+Na⁺ matrix) m/e: ([M+Na]⁺)691.5504 (38.5%), calcd. 691.5502.

HCl salt of compound 2: Compound 2 was dissolved in a minimum amount ofCH₂Cl₂ and excess HCl in ether was added. Removal of the solvent andexcess HCl gave a noncrystalline white powder. 1H NMR (methanol-d₄/15%(CDCl₃, 300 MHz) δ 7.60-7.59 (m, 2H), 7.50-7.47 (m, 3H), 4.82 (bs, 10H),4.43 (bs, 1H), 4.32 (bs, 1H), 3.85-3.79 (m, 1H), 3.75-3.68 (m, 1H), 3.64(t, J=5.74 Hz, 2H), 3.57 (bs, 1H), 3.36-3.28 (m, 2H), 3.25-3.00 (seriesof multiplets, 10H), 2.82 (s, 3H), 2.14-1.68 (series of multiplets,19H), 1.65-1.15 (series of multiplets, 11H), 0.98 (d, J=6.6 Hz, 3H),0.95 (s, 3H), 0.72 (s, 3H); ¹³C NMR (methanol-d4/15% (CDCl₃, 75 MHz) δ132.21, 131.10, 130.58, 130.28, 81.96, 80.72, 77.60, 66.84, 66.58,66.12, 61.03, 57.60, 44.16, 42.77, 40.62, 39.57, 39.43, 36.28, 36.03,35.96, 35.78, 33.65, 29.48, 29.27, 29.11, 29.01, 28.61, 28.56, 28.35,24.25, 23.56, 23.30, 21.17, 18.64, 12.90.

Compound 4: A suspension of 1 (79.1 mg, 0.126 mmol) andaminoiminomethanesulfonic acid (50.15 mg, 0.404 mmol) in methanol andchloroform was stirred at room temperature for 24 hours, and thesuspension became clear. An ether solution of HCl (1 M, 1 mL) was addedfollowed by the removal of solvent with N₂ flow. The residue wasdissolved in H₂O (5 mL) followed by the addition of 20% aqueous NaOH(0.5 mL). The resulting cloudy mixture was extracted with CH₂Cl₂ (4×5mL). The combined extracts were dried over anhydrous Na₂SO₄. Removal ofsolvent gave the desired product (90 mg, 95%) as white powder. m.p.111-112° C. IR (neat) 3316, 2937, 1667, 1650, 1556, 1454, 1348, 1102cm⁻¹; H NMR (5% methanol-d4/CDCl₃, 300 MHz) δ 7.26-7.22 (m, 5H), 4.37(bs, 3H), 3.71-3.51 (series of multiplets, 5H), 3.44 (s, 2H), 3.39-3.10(series of multiplets, 10H), 2.27 (t, J=6.83 Hz, 2H), 2.13 (s, 3H),2.02-0.94 (series of multiplets, 33H), 0.85 (d, J=5.62 Hz, 3H), 0.84 (s,3H), 0.61 (s, 3H); ¹³C NMR (5% methanol-d4/CDCl₃, 75 MHz) δ 158.54,158.48, 158.43, 138.27, 129.47, 128.32, 127.19, 81.89, 80.30, 77.34,69.02, 68.46, 67.21, 62.36, 58.00, 47.36, 46.18, 43.26, 43.00, 42.73,42.18, 41.48, 39.32, 35.55, 34.97, 34.89, 34.67, 33.63, 28.93, 28.28,27.53, 27.16, 23.96, 23.28, 23.16, 22.77, 18.36, 12.58; HRFAB-MS(thioglycerol+Na⁺ matrix) m/e: ([M+H]⁺) 753.5858 (100%), calcd.753.5867.

HCl salt of compound 4: Compound 4 was dissolved in minimum amount ofCH₂Cl₂ and MeOH followed by addition of excess HCl in ether. The solventwas removed by N₂ flow, and the residue was subjected to high vacuumovernight. The desired product was obtained as noncrystalline whitepowder. ¹H NMR (methanol-d4/20% (CDCl₃, 300 MHz) δ 7.58 (bs, 2H),7.50-7.48 (m, 3H), 4.76 (bs, 13H), 4.45 (d, J=12.9 Hz, 1H), 4.27 (dd,1H, J=12.9, 5.4 Hz), 3.82-3.00 (series of multiplets, 17H), 2.81-2.80(m, 3H), 2.20-1.02 (series of multiplets, 27H), 0.98 (d, J=6.59 Hz, 3H),0.95 (s, 3H), 0.72 (s, 3H); ¹³C NMR (methanol-d4/20% CDCl₃, 75 MHz) δ158.88, 158.72, 132.00, 131.96, 130.98, 130.15, 82.51, 81.07, 78.05,68.50, 68.02, 67.94, 67.10, 60.87, 60.53, 57.38, 47.16, 46.91, 43.91,43.11, 43.01, 42.91, 42.55, 40.28, 39.88, 39.95, 35.90, 35.73, 35.64,33.53, 29.18, 28.35, 27.99, 24.02, 23.30, 21.35, 18.52, 18.44, 13.06.

Compound 5: A suspension of 2 (113 mg, 0.169 mmol) andaminoiminomethanesulfonic acid (67.1 mg, 0.541 mmol) in methanol andchloroform was stirred at room temperature for 24 hours. HCl in ether (1M, 1 mL) was added followed by the removal of solvent with N₂ flow. Theresidue was subject to high vacuum overnight and dissolved in H₂O (5 mL)followed by the addition of 20% NaOH solution (1.0 mL). The resultingmixture was extracted with CH₂Cl₂ (5×5 mL). The combined extracts weredried over anhydrous Na₂SO₄. Removal of solvent gave desired the product(90 mg, 95% yield) as a white solid. m.p. 102-104° C. IR (neat) 3332,3155, 2939, 2863, 1667, 1651, 1558, 1456, 1350, 1100 cm⁻¹; ¹H NMR (5%methanol-d4/CDCl₃, 300 MHz) δ 7.35-7.24 (m, 5H), 3.75-3.64 (m, 1H), 3.57(bs, 5H), 3.50 (s, 2H), 3.53-3.46 (m, 1H), 3.40-3.10 (series ofmultiplets, 14H), 2.34 (t, J=7.31 Hz, 2H), 2.19 (s, 3H), 2.13-0.96(series of multiplets, 36H), 0.91 (bs, 6H), 0.66 (s, 3H); ¹³C NMR (5%methanol-d4/CDCl₃, 75 MHz) δ 157.49, 157.31, 157.23, 138.20, 129.52,128.34, 127.23, 81.17, 79.19, 76.42, 65.63, 65.03, 64.70, 62.36, 58.02,47.23, 46.24, 42.89, 42.18, 41.45, 39.45, 39.40, 39.30, 38.71, 35.61,35.55, 35.02, 34.82, 33.69, 29.87, 29.59, 29.42, 28.84, 27.96, 27.56,23.95, 23.40, 22.82, 22.64, 18.28, 12.54; HRFAB-MS (thioglycerol+Na⁺matrix) m/e: ([M+H]⁺) 795.6356 (84.3%), calcd. 795.6337.

HCl salt of compound 5: Compound 5 was dissolved in minimum amount ofCH₂Cl₂ and MeOH followed by the addition of excess HCl in ether. Thesolvent and excess HCl were removed by N₂ flow and the residue wassubject to high vacuum overnight. The desired product was obtained asnoncrystalline white powder. ¹H NMR (methanol-d4/10% CDCl₃, 300 MHz) δ7.62-7.54 (m, 2H), 7.48-7.44 (m, 3H), 4.84 (bs, 16H), 4.46 (d, J=12.7Hz, 1H), 4.26 (dd, J=12.7, 3.42 Hz, 1H), 3.78-3.56 (series ofmultiplets, 5H), 3.38-3.05 (series of multiplets, 13H), 2.80 (d, 3H),2.19-2.04 (m, 3H), 2.02-1.04 (series of multiplets, 30H), 0.98 (d,J=6.35 Hz, 3H), 0.95 (s, 3H), 0.72 (s, 3H); ¹³C NMR (methanol-d4/10%CDCl₃, 75 MHz) δ 158.75, 158.67, 132.32, 131.24, 130.83, 130.43, 82.49,81.02, 77.60, 66.47, 65.93, 61.19, 60.85, 57.69, 47.79, 47.60, 44.29,43.07, 40.86, 40.42, 40.19, 40.09, 39.76, 36.68, 36.50, 36.15, 35.94,33.91, 30.75, 30.46, 29.74, 29.33, 28.71, 24.41, 24.03, 23.38, 22.21,22.16, 18.59, 18.52, 13.09.

Compound CSA-26 was synthesized according to Scheme 1 and Example 1using 7-deoxycholic acid in place of cholic acid and methyl cholate.

Example 2

This example includes a description of one or more exemplary synthesticprocedures for obtaining Compounds 3, 28 and 29.

Compound 28: A suspension of 19 (0.641 g, 0.614 mmol) and KCN (0.40 g,6.14 mmol) in anhydrous DMSO (5 mL) was stirred under N₂ at 80° C.overnight followed by the addition of H₂O (50 mL). The aqueous mixturewas extracted with EtOAc (4×20 mL). The combined extracts were washedwith brine once, dried over anhydrous Na₂SO₄ and concentrated in vacuo.The residue was dissolved in CH₂Cl₂ (3 mL) and MeOH (3 mL) and catalyticamount of p-toluenesulfonic acid (5.84 mg, 0.03 mmol) was added. Thesolution was stirred at room temperature for 3 hours before theintroduction of saturated NaHCO₃ solution (10 mL). After the addition ofbrine (60 mL), the mixture was extracted with EtOAc (4×30 mL). Thecombined extracts were washed with brine once and dried over anhydrousNa₂SO₄ and concentrated. The residue afforded the desired product (0.342g, 92% yield) as pale yellowish oil after column chromatography (silicagel, EtOAc/hexanes 2:1). IR (neat) 3479, 2936, 2864, 2249, 1456, 1445,1366, 1348, 1108 cm⁻¹; ¹H NMR (CDCl₃, 300 MHz) δ 3.76-3.53 (m, 7H),3.32-3.06 (series of multiplets, 4H), 2.57-2.46 (m, 6H), 2.13-1.00(series of multiplets, 31H), 0.93 (d, J=6.35 Hz, 3H), 0.90 (s, 3H), 0.67(s, 3H); ¹³C NMR (CDCl₃, 75 MHz) δ 119.91, 119.89, 80.75, 79.65, 76.29,65.83, 65.37, 65.19, 63.63, 46.57, 46.44, 42.77, 41.79, 39.71, 35.63,35.26, 35.02, 32.00, 29.46, 29.03, 27.96, 27.74, 26.64, 26.42, 26.12,23.56, 22.98, 22.95, 18.24, 14.65, 14.54, 14.30, 12.60; HRFAB-MS(thioglycerol+Na⁺ matrix) m/e: ([M+Na]⁺) 618.4247 (67.8%), calcd.618.4247.

Compound 29: To a solution of 28 (0.34 g, 0.57 mmol) in dry CH₂Cl₂ (15mL) under N₂ at 0° C. was added NEt₃ (119.5 μL, 0.857 mmol) followed bythe addition of mesyl chloride (53.1.mu.L, 0.686 mmol). The mixture wasallowed to stir at 0° C. for 30 minutes before the addition of H₂O (6mL). After the introduction of brine (60 mL), the aqueous mixture wasextracted with EtOAc (4×20 mL). The combined extracts were washed withbrine once, dried over anhydrous Na₂SO₄ and concentrated. To the residuewas added N-benzylmethyl amine (0.5 mL) and the mixture was stirredunder N₂ at 80° C. overnight. Excess N-benzylmethylamine was removed invacuo and the residue was subject to column chromatography (silica gel,EtOAc/hexanes 2:1 followed by EtOAc) to afford product (0.35 g, 88%yield) as a pale yellow oil. IR (neat) 2940, 2863, 2785, 2249, 1469,1453, 1366, 1348, 1108 cm⁻¹; ¹H NMR (CDCl₃, 300 MHz) δ 7.34-7.21 (m,5H), 3.76-3.69 (m, 1H), 3.64-3.50 (m, 4H), 3.48 (s, 2H), 3.31-3.05(series of multiplets, 4H), 2.52-2.46 (m, 6H), 2.33 (t, J=7.32H, 2 Hz),2.18 (s, 3H), 2.13-0.95 (series of multiplets, 30H), 0.91 (d, J=6.80H, 3Hz), 0.90 (s, 3H), 0.66 (s, 3H); ¹³C NMR (CDCl₃, 75 MHz) δ 139.37,129.17, 128.26, 126.93, 119.96, 119.91, 80.73, 79.59, 76.26, 65.79,65.35, 65.13, 62.47, 58.25, 46.74, 46.40, 42.72, 42.38, 41.76, 39.68,35.78, 35.22, 34.98, 33.79, 28.99, 27.92, 27.71, 26.63, 26.38, 26.09,24.21, 23.54, 22.96, 22.90, 18.28, 14.62, 14.51, 14.26, 12.58; HRFAB-MS(thioglycerol+Na⁺ matrix) m/e: ([M+H]⁺) 699.5226 (100%), calcd.699.5213.

Compound 3: A solution of 29 (0.074 g, 0.106 mmol) in anhydrous THF (10mL) was added dropwise to a mixture of AlCl₃ (0.1414 g, 1.06 mmol) andLiAlH₄ (0.041 g, 1.06 mmol) in dry THF (10 mL). The suspension wasstirred for 24 hours followed by the addition of 20% NaOH aqueoussolution (2 mL) at ice-bath temperature. Anhydrous Na₂SO₄ was added tothe aqueous slurry. The solution was filtered and the precipitate washedtwice with THF. After removal of solvent, the residue was subject tocolumn chromatography (silica gel, MeOH/CH₂Cl₂ 1:1 followed byMeOH/CH₂Cl₂/NH₃.H₂O 4:4:1) to afford the desired product (0.038 g, 50%yield) as a clear oil. IR (neat) 3362, 2935, 2863, 2782, 1651, 1574,1568, 1557, 1471, 1455, 1103 cm⁻¹; ¹H NMR (CDCl₃, 300 MHz) δ 7.32-7.22(m, 5H), 3.60-3.02 (series of broad multiplets, 18H), 2.90-2.70 (m, 5H),2.33 (t, J=7.20 Hz, 2H), 2.24-2.04 (m, 3H), 2.18 (s, 3H), 1.96-0.96(series of multiplets, 30H), 0.90 (d, J=7.57 Hz, 3H), 0.89 (s, 3H), 0.64(s, 3H); ¹³C NMR (CDCl₃, 75 MHz) δ 139.44, 129.24, 128.31, 126.97,80.63, 79.65, 75.97, 68.44, 68.00, 67.96, 62.54, 58.40, 46.77, 46.30,42.73, 42.43, 42.07, 41.92, 41.74, 41.72, 39.81, 35.82, 35.48, 35.07,33.84, 31.04, 30.30, 30.10, 29.03, 28.11, 27.82, 27.81, 27.74, 27.67,27.64, 24.31, 23.50, 23.04, 22.93, 18.22, 12.63; HRFAB-MS(thioglycerol+Na⁺ matrix) m/e: ([M+H]⁺) 711.6139 (100%), calcd.711.6152; ([M+Na]⁺) 733.5974 (46.1%), calcd. 733.5972.

Example 3

This example includes a description of one or more exemplary synthesticprocedures for obtaining Compounds 6, 7 and 30-33.

Compound 30: Cholic acid (3.0 g, 7.3 mmol) was dissolved in CH₂Cl₂ (50mL) and methanol (5 mL). Dicyclohexylcarbodiimide (DCC) (1.8 g, 8.8mmol) was added followed by N-hydroxysuccinimide (about 100 mg) andbenzylmethylamine (1.1 g, 8.8 mmol). The mixture was stirred for 2hours, then filtered. The filtrate was concentrated and chromatographed(SiO₂, 3% MeOH in CH₂Cl₂) to give 3.0 g of a white solid (81% yield).m.p. 184-186° C.; IR (neat) 3325, 2984, 1678 cm⁻¹; ¹H NMR (CDCl₃, 200MHz) δ 7.21 (m, 5H), 4.51 (m, 2H), 3.87 (m, 1H), 3.74 (m, 2H), 3.36 (m,2H), 2.84 (s, 3H), 2.48-0.92 (series of multiplets, 28H), 0.80 (s, 3H),0.58 (d, J=6.5 Hz, 3H); ¹³C NMR (CDCl₃, 50 MHz) δ 174.30, 173.94,137.36, 136.63, 128.81, 128.46, 127.85; 127.50, 127.18, 126.28, 72.96,71.76, 68.35, 53.39, 50.65, 48.77, 46.91, 46.33, 41.44, 39.36, 39.18,35.76, 35.27, 34.76, 33.87, 31.54, 34.19, 31.07, 30.45, 28.11, 27.63,26.14, 25.59, 24.92, 23.26, 17.51, 12.41; FAB-MS (thioglycerol+Na⁺matrix) m/e: ([M+H]⁺) 512 (100%), calcd. 512.

Compound 31: Compound 30 (2.4 g, 4.7 mmol) was added to a suspension ofLiAlH₄ (0.18 g, 4.7 mmol) in THF (50 mL). The mixture was refluxed for24 hours, then cooled to 0° C. An aqueous solution of Na₂SO₄ wascarefully added until the grey color of the mixture dissipated. Thesalts were filtered out, and the filtrate was concentrated in vacuo toyield 2.1 g of a white solid (88%). The product proved to be ofsufficient purity for further reactions. m.p. 70-73° C.; IR (neat) 3380,2983, 1502 cm⁻¹; ¹H NMR (CDCl₃, 300 MHz) δ 7.23 (m, 5H), 3.98 (bs, 2H),3.81 (m, 3H), 3.43 (m, 3H), 2.74 (m, 2H), 2.33 (m, 3H), 2.25 (s, 3H),2.10-0.90 (series of multiplets, 24H), 0.98 (s, 3H), 0.78 (s, 3H); 13CNMR (CDCl₃, 75 MHz) δ 135.72, 129.63, 128.21, 128.13, 125.28, 72.91,71.63, 62.05, 60.80, 56.79, 47.00, 46.23, 41.44, 40.81, 39.41, 35.42,35.24, 34.63, 34.02, 33.22, 31.73, 30.17, 29.33, 29.16, 28.02, 27.49,26.17, 25.55, 23.10, 22.48, 22.33, 17.54, 12.65; FAB-MS (thioglycerolmatrix) m/e: ([M+H]⁺) 498 (100%), calcd. 498.

Compound 32: Compound 31 (0.36 g, 0.72 mmol) was dissolved in CH₂Cl₂ (15mL) and Bocglycine (0.51 g, 2.89 mmol), DCC (0.67 g, 3.24 mmol) anddimethylaminopyridine (DMAP) (about 100 mg) were added. The mixture wasstirred under N₂ for 4 hours then filtered. After concentration andchromatography (SiO₂, 5% MeOH in CH₂Cl₂), the product was obtained as a0.47 g of a clear glass (68%). ¹H NMR (CDCl₃, 300 MHz) δ 7.30 (m, 5H),5.19 (bs, 1H), 5.09 (bs, 3H), 5.01 (bs, 1H), 4.75 (m, 1H), 4.06-3.89 (m,6H), 2.33 (m, 2H), 2.19 (s, 3H) 2.05-1.01 (series of multiplets, 26H),1.47 (s, 9H), 1.45 (s, 18H), 0.92 (s, 3H), 0.80 (d, J=6.4 Hz, 3H), 0.72(s, 3H). ¹³C NMR (CDCl₃, 75 MHz) δ 170.01, 169.86, 169.69, 155.72,155.55, 139.90, 129.05, 128.17, 126.88, 79.86, 76.53, 75.09, 72.09, 62,35, 57.88, 47.78, 45.23, 43.12, 42.79, 42.16, 40.81, 37.94, 35.51,34.69, 34.57, 34.36, 33.30, 31.31, 29.66, 28.80, 28.34, 27.22, 26.76,25.61, 24.02, 22.83, 22.47, 17.93, 12.19; FAB-MS (thioglycerol matrix)m/e: ([M+H]⁺) 970 (100%), calcd. 970.

Compound 33: Compound 31 (0.39 g, 0.79 mmol) was dissolved in CH₂Cl₂ (15mL) and Boc-β-alanine (0.60 g, 3.17 mmol), DCC (0.73 g, 3.56 mmol) anddimethylaminopyridine (DMAP) (about 100 mg) were added. The mixture wasstirred under N₂ for 6 hours then filtered. After concentration andchromatography (SiO₂, 5% MeOH in CH₂Cl₂), the product was obtained as a0.58 g of a clear glass (72%). IR (neat) 3400, 2980, 1705, 1510 cm⁻¹; ¹HNMR (CDCl₃, 300 MHz) δ 7.27 (m, 5H), 5.12 (bs, 4H), 4.93 (bs, 1H), 4.71(m, 1H), 3.40 (m, 12H), 2.59-2.48 (m, 6H), 2.28 (m, 2H), 2.17 (s, 3H),2.05-1.01 (series of multiplets, 26H), 1.40 (s, 27H), 0.90 (s, 3H), 0.77(d, J=6.1 Hz, 3H), 0.70 (s, 3H). ¹³C NMR (CDCl₃, 75 MHz) δ 171.85,171.50, 171.44, 155.73, 138.62, 129.02, 128.09, 126.87, 79.18, 75.53,74.00, 70.91, 62.20, 57.67, 47.84, 44.99, 43.28, 41.98, 40.73, 37.67,36.12, 34.94, 34.65, 34.47, 34.20, 33.29, 31.23, 29.57, 28.74, 28.31,28.02, 27.86, 27.12, 26.73, 25.46, 24.86, 23.95, 22.77, 22.39, 17.91,12.14; HRFAB-MS (thioglycerol+Na⁺ matrix) m/e: ([M+H]⁺) 1011.6619(100%), calcd. 1011.6634.

Compound 6: Compound 32 (0.15 g, 0.15 mmol) was stirred with excess 4NHCl in dioxane for 40 minutes. The dioxane and HCl were removed in vacuoleaving 0.12 g of a clear glass (about 100%). ¹H NMR (CD₃OD, 300 MHz) δ7.62 (bs, 2H), 7.48 (bs, 3H), 5.30 (bs, 1H), 5.11 (bs, 1H), 4.72 (bs(1H), 4.46 (m, 1H), 4.32 (m, 1H) 4.05-3.91 (m, 4H), 3.10 (m, 2H), 2.81(s, 3H), 2.15-1.13 (series of multiplets, 25H), 1.00 (s, 3H), 0.91 (bs,3H), 0.82 (s, 3H). ¹³C NMR (CD₃OD, 125 MHz) δ 166.86, 166.50, 131.09,130.18, 129.17, 128.55, 76.60, 75.43, 72.61, 72.04, 70.40, 66.22, 60.07,58.00, 57.90, 54.89, 54.76, 46.44, 44.64, 43.39, 42.22, 38.56, 36.78,34.14, 33.92, 33.84, 31.82, 30.54, 29.67, 28.79, 27.96, 26.79, 26.00,24.99, 23.14, 22.05, 21.82, 19.91, 17.27, 11.60; HRFAB-MS(thioglycerol+Na⁺ matrix) m/e: ([M-4 Cl-3H]⁺) 669.4576 (100%), calcd.669.4591.

Compound 7: Compound 33 (0.20 g, 0.20 mmol) was stirred with excess 4NHCl in dioxane for 40 minutes. The dioxane and HCl were removed in vacuoleaving 0.12 g of a clear glass (about 100%). ¹H NMR (CD₃OD, 500 MHz) δ7.58 (bs, 2H), 7.49 (bs, 3H), 5.21 (bs, 1H), 5.02 (bs, 1H), 4.64 (m,1H), 4.44 (m, 1H), 4.28 (m, 1H), 3.30-2.84 (m, 14H), 2.80 (s, 3H),2.11-1.09 (series of multiplets, 25H), 0.99 (s, 3 H), 0.89 (d, J=4.1 Hz,3H), 0.80 (s, 3H); ¹³C NMR (CD₃ OD, 125 MHz) δ 171.92, 171.56, 171.49,132.44, 131.32, 131.02, 130.51, 78.13, 76.61, 61.45, 57.94, 46.67,44.80, 42.36, 40.85, 39.33, 37.03, 36.89, 36.12, 36.09, 35.79, 35.63,33.81, 33.10, 32.92, 32.43, 30.28, 28.43, 28.04, 26.65, 24.02, 22.86,21.98, 18.70, 12.68; HRFAB-MS (thioglycerol+Na⁺ matrix) m/e: ([M-4C1-3H]⁺) 711.5069 (43%), calcd. 711.5061.

Example 4

This example includes a description of one or more exemplary synthesticprocedures for obtaining Compounds 8, CSA-7, CSA-8 and 34-40.

Compound 34: Diisopropyl azodicarboxylate (DIAD) (1.20 mL, 6.08 mmol)was added to triphenylphosphine (1.60 g, 6.08 mmol) in THF (100 mL) at0° C. and was stirred for half an hour during which time the yellowsolution became a paste. Compound 14 (2.58 g, 4.06 mmol) andp-nitrobenzoic acid (0.81 g, 4.87 mmol) were dissolved in THF (50 mL)and added to the paste. The resulted mixture was stirred at ambienttemperature overnight. Water (100 mL) was added and the mixture was madeslightly basic by adding NaHCO₃ solution followed by extraction withEtOAc (3×50 mL). The combined extracts were washed with brine once anddried over anhydrous Na₂SO₄. The desired product (2.72 g, 85% yield) wasobtained as white powder after SiO₂ chromatography (Et₂O/hexanes 1:2).m.p. 207-209° C.; IR (KBr) 3434, 3056, 2940, 2868, 1722, 1608, 1529,1489, 1448, 1345 cm⁻¹; ¹H NMR (CDCl₃, 300 MHz) δ 8.30-8.26 (m, 2H),8.21-8.16 (m, 2H), 7.46-7.42 (m, 6H), 7.31-7.18 (m, 9H) 5.33 (bs, 1H),4.02 (bs, 1H), 3.90 (bs, 1H), 3.09-2.97 (m, 2H), 2.68 (td, J=14.95, 2.56Hz, 1H), 2.29-2.19 (m, 1H), 2.07-1.06 (series of multiplets, 24H), 1.01(s, 3H), 0.98 (d, J=6.6 Hz, 3H), 0.70 (s, 3H); ¹³C NMR (CDCl₃, 75 MHz) δ164.21, 150.56, 144.70, 136.79, 130.77, 128.88, 127.86, 126.98, 123.70,86.47, 73.24, 73.00, 68.70, 64.22, 47.79, 46.79, 42.15, 39.76, 37.47,35.52, 35.34, 34.23, 33.79, 32.46, 31.12, 28.74, 27.71, 26.85, 26.30,25.16, 23.41, 17.98, 12.77; HRFAB-MS (thioglycerol+Na⁺ matrix) m/e:([M+Na]⁺) 808.4203 (53.8%), calcd. 808.4189. Nitrobenzoate (2.75 g, 3.5mmol) was dissolved in CH₂Cl₂ (40 mL) and MeOH (20 mL) and 20% aqueousNaOH (5 mL) were added. The mixture was heated up to 60° C. for 24hours. Water (100 mL) was introduced and extracted with EtOAc. Thecombined extracts were washed with brine and dried over anhydrousNa₂SO₄. The desired product (1.89 g, 85% yield) was obtained as whitesolid after SiO₂ chromatography (3% MeOH in CH₂Cl₂ as eluent). m.p.105-106° C.; IR (KBr) 3429, 3057, 2936, 1596, 1489, 1447, 1376, 1265,1034, 704 cm⁻¹; ¹H NMR (CDCl₃, 300 MHz) δ 7.46-7.42 (m, 6H), 7.32-7.19(m, 9H), 4.06 (bs, 1H), 3.99 (bs, 1H), 3.86 (bd, J=2.44 Hz, 1H),3.09-2.97 (m, 2H), 2.47 (td, J=14.03, 2.44 Hz, 1H), 2.20-2.11 (m, 1H),2.04-1.04 (series of multiplets, 25H), 0.97 (d, J=6.59 Hz, 3H), 0.94 (s,3H), 0.68 (s, 3H); ¹³C NMR (CDCl₃, 75 MHz) δ 144.70, 128.88, 127.86,126.97, 86.45, 73.31, 68.84, 67.10, 64.23, 47.71, 46.74, 42.10, 39.70,36.73, 36.73, 36.15, 35.53, 35.45, 34.45, 32.46, 29.93, 28.67, 27.86,27.71, 26.87, 26.04, 23.43, 23.16, 17.94, 12.75; HRFAB-MS(thioglycerol+Na⁺ matrix) m/e: ([M+Na]⁺) 659.4064 (100%), calcd.659.4076.

Compound 35: To a round-bottom flask were added 34 (2.0 g, 3.14 mmol),NaH (60% in mineral oil, 3.8 g, 31.4 mmol) and THF (150 mL). Thesuspension was refluxed for 2 hours followed by the addition of allylbromide (2.72 mL, 31.4 mL). After refluxing for 28 hours, another 10 eq.of NaH and allyl bromide were added. After 72 hours, another 10 eq. ofNaH and allyl bromide were added. After 115 hours, TLC showed almost nostarting material or intermediates. Water (100 mL) was added to thesuspension carefully, followed by extraction with EtOAc (5×50 mL). Thecombined extracts were washed with brine and dried over anhydrousNa₂SO₄. The desired product (1.81 g, 79% yield) was obtained as ayellowish glass after SiO₂ chromatography (5% EtOAc/hexanes). IR (neat)3060, 3020, 2938, 2865, 1645, 1596, 1490, 1448, 1376, 1076, 705 cm⁻¹; ¹HNMR (CDCl₃, 300 MHz) δ 7.46-7.42 (m, 6H), 7.31-7.18 (m, 9H), 6.06-5.85(m, 3H), 5.35-5.20 (m, 3H), 5.15-5.06 (m, 3H), 4.10-4.00 (m, 2H),3.93-3.90 (m, 2H), 3.85-3.79 (ddt, J=13.01, 4.88, 1.59 Hz, 1H),3.73-3.66 (ddt, J=13.01, 5.38, 1.46 Hz, 1H), 3.58 (bs, 1H), 3.54 (bs,1H), 3.32 (d, J=2.93 Hz, 1H), 3.07-2.96 (m, 2H), 2.36 (td, J=13.67, 2.68Hz, 1H), 2.24-2.10 (m, 2H), 2.03-1.94 (m, 1H), 1.87-0.86 (series ofmultiplets, 20H), 0.91 (s, 3H), 0.90 (d, J=6.83 Hz, 3H), 0.64 (s, 3H);¹³C NMR (CDCl₃, 75 MHz) δ 144.77, 136.29, 136.21, 136.13, 128.90,127.86, 126.94, 116.13, 115.51, 115.42, 86.44, 81.11, 75.65, 73.92,69.40, 68.81, 64.43, 46.68, 46.54, 42.93, 39.93, 36.98, 35.66, 35.14,35.14, 32.83, 32.54, 30.48, 28.51, 27.72, 27.64, 26.82, 24.79, 23.65,23.43, 23.40, 18.07, 12.80; HRFAB-MS (thioglycerol+Na⁺ matrix) m/e:([M+H]⁺) 757.5185 (12.9%), calcd. 757.5196.

Compound 36: Ozone was bubbled through a solution of 35 (0.551 g, 0.729mmol) in CH₂Cl₂ (40 mL) and MeOH (20 mL) at −78° C. until the solutionturned a deep blue. Excess ozone was blown off with oxygen.Methylsulfide (1 mL) was added followed by the addition of NaBH₄ (0.22g, 5.80 mmol) in 5% NaOH solution and methanol. The resulted mixture wasstirred overnight at room temperature and washed with brine. The brinewas then extracted with EtOAc (3×20 mL). The combined extracts weredried over Na₂SO₄. The desired product (0.36 g, 65% yield) was obtainedas a colorless glass after SiO₂ chromatography (5% MeOH/CH₂Cl₂). IR(neat) 3396, 3056, 2927, 1596, 1492, 1462, 1448, 1379, 1347, 1264, 1071cm⁻¹; ¹H NMR (CDCl₃, 300 MHz) δ 7.46-7.42 (m, 6H), 7.32-7.18 (m, 9H),3.77-3.57 (series of multiplets, 10H), 3.48-3.44 (m, 2H), 3.36-3.30 (m,2H), 3.26-3.20 (m, 1H), 3.04-2.99 (m, 2H), 2.37-0.95 (series ofmultiplets, 27H), 0.92 (s, 3H), 0.91 (d, J=6.59 Hz, 3H), 0.67 (s, 3H);¹³C NMR (CDCl₃, 75 MHz) δ 144.69, 128.87, 127.84, 126.94, 86.44, 81.05,76.86, 74.65, 69.91, 69.22, 68.77, 64.24, 62.44, 62.42, 62.26, 46.92,46.54, 42.87, 39.73, 36.86, 35.52, 35.13, 32.82, 32.54, 30.36, 28.71,27.61, 27.44, 26.79, 24.82, 23.51, 23.38, 23.31, 18.28, 12.74; HRFAB-MS(thioglycerol+Na⁺ matrix) m/e: ([M+Na]⁺) 791.4844 (96.4%), calcd.791.4863.

Compound 37: NEt₃ (0.23 mL, 1.66 mmol) was added to a solution of 36(0.364 g, 0.47 mmol) in dry CH₂Cl₂ (30 mL) at 0° C. under N₂ followed bythe introduction of mesyl chloride (0.12 mL, 1.56 mmol). The mixture wasstirred for 10 minutes and H₂O (10 mL) added to quench the reaction,followed by extraction with EtOAc (3×30 mL). The combined extracts werewashed with brine and dried over anhydrous Na₂SO₄. SiO₂ chromatography(EtOAc/hexanes 1:1) gave the desired product (0.411 g, 86% yield) aswhite glass. IR (neat) 3058, 3029, 2939, 2868, 1491, 1461, 1448, 1349,1175, 1109, 1019 cm⁻¹; ¹H NMR (CDCl₃, 300 MHz) δ 7.46-7.42 (m, 6H),7.31-7.19 (m, 9H), 4.35-4.26 (m, 6H), 3.84-3.74 (m, 2H), 3.64-3.56 (m,4H), 3.49-3.34 (m, 3H), 3.06 (s, 3H), 3.04 (s, 3H), 3.02 (s, 3H),3.09-2.95 (m, 2H), 2.28 (bt, J=14.89 Hz, 1H), 2.09-0.86 (series ofmultiplets, 21H), 0.92 (s, 3H), 0.90 (d, J=6.78 Hz, 3H), 0.66 (s, 3H);¹³C NMR (CDCl₃, 75 MHz) δ 144.66, 128.86, 127.86, 126.97, 86.46, 81.28,77.18, 75.00, 70.14, 69.89, 69.13, 66.49, 65.85, 65.72, 64.22, 47.06,46.35, 42.77, 39.58, 37.81, 37.64, 37.55, 36.75, 35.48, 35.02, 32.59,32.52, 30.27, 28.43, 27.56, 27.52, 26.92, 24.62, 23.34, 23.25, 23.10,18.24, 12.64; HRFAB-MS (thioglycerol+Na⁺ matrix) m/e: ([M+Na]⁺)1025.4207 (100%), calcd. 1025.4189.

Compound 38: The suspension of 37 (0.227 g, 0.227 mmol) and NaN₃ (0.147g, 2.27 mmol) in dry DMSO (5 mL) was stirred at 80° C. overnight,diluted with H₂O (50 mL) and extracted with EtOAc (3×20 mL). Theextracts were washed with brine once and dried over anhydrous Na₂SO₄.SiO₂ chromatography (EtOAc/hexanes 1:8) afforded the desired product(0.153 g, 80% yield) as a yellow oil. IR (neat) 2929, 2866, 2105, 1490,1466, 1448, 1107, 705 cm⁻¹; ¹H NMR (CDCl₃, 300 MHz) δ 7.46-7.42 (m, 6H),7.32-7.19 (m, 9H), 3.80-3.74 (m, 1H), 3.70-3.55 (series of multiplets,5H), 3.41-3.19 (series of multiplets, 9H), 3.04-2.98 (m, 2H), 2.41 (td,J=13.1, 2.44 Hz, 1H), 2.29-2.14 (m, 2H), 2.04-0.86 (series ofmultiplets, 20H), 0.93 (s, 3H), 0.91 (d, J=6.60 Hz, 3H), 0.66 (s, 3H);¹³C NMR (CDCl₃, 75 MHz) δ 144.78, 128.93, 127.87, 126.96, 86.46, 81.30,77.16, 75.21, 67.99, 67.44, 67.03, 64.41, 51.64, 51.57, 51.33, 46.71,46.30, 42.35, 39.75, 36.72, 35.64, 35.20, 32.52, 32.42, 30.17, 28.63,27.80, 27.22, 26.90, 24.80, 23.55, 23.30, 23.24, 18.23, 12.65; HRFAB-MS(thioglycerol+Na⁺ matrix) m/e: ([M+Na]⁺) 866.5049 (96.9%), calcd.866.5057.

Compound 39: p-Toluenesulfonic acid (1.72 mg) was added into thesolution of 38 (0.153 g, 0.18 mmol) in CH₂Cl₂ (5 mL) and MeOH (5 mL),and the mixture was stirred for 2.5 hours. Saturated NaHCO₃ solution (5mL) was introduced followed by the introduction of brine (30 mL). Theaqueous mixture was extracted with EtOAc and the combined extractswashed with brine and dried over Na₂SO₄. The desired product (0.10 g,92% yield) was obtained as a pale yellowish oil after SiO₂chromatography (EtOAc/hexanes 1:3). IR (neat) 3426, 2926, 2104, 1467,1441, 1347, 1107 cm⁻¹; ¹H NMR (CDCl₃, 300 MHz) δ 3.81-3.74 (m, 1H),3.71-3.54 (m, 7H), 3.41-3.19 (m, 9H), 2.41 (td, J=13.61, 2.32 Hz, 1H),2.30-2.14 (m, 2H), 2.07-1.98 (m, 1H), 1.94-0.95 (series of multiplets,21H), 0.95 (d, J=6.35 Hz, 3H), 0.93 (s, 3H), 0.69 (s, 3H); ¹³C NMR(CDCl₃, 75 MHz) S 81.22, 77.08, 75.13, 67.94, 67.36, 66.97, 63.76,51.59, 51.51, 51.26, 46.51, 46.24, 42.31, 39.68, 36.64, 35.58, 35.12,32.34, 31.92, 30.11, 29.55, 28.54, 27.82, 27.16, 24.75, 23.47, 23.23,23.18, 18.15, 12.56; HRFAB-MS (thioglycerol+Na⁺ matrix) m/e: ([M+Na]⁺)624.3966 (54.9%), calcd. 624.3962.

Compound 40: To a solution of 39 (0.10 g, 0.166 mmol) in CH₂Cl₂ (8 mL)at 0° C. was added NEt₃ (34.8 μL, 0.25 mmol) under N₂ followed by theintroduction of mesyl chloride (15.5.mu.L, 0.199 mmol). The mixture wasstirred 15 minutes. Addition of H₂O (3 mL) and brine (20 mL) wasfollowed by extraction with EtOAc (4×10 mL). The combined extracts werewashed with brine once and dried over Na₂SO₄. After removal of solvent,the residue was mixed with N-benzylmethylamine (0.5 mL) and heated to80° C. under N₂ overnight. Excess N-benzyl methylamine was removed invacuo and the residue was subjected to SiO₂ chromatography(EtOAc/hexanes 1:4) to give the product (0.109 g, 93% yield) as a yellowoil. IR (neat) 2936, 2784, 2103, 1467, 1442, 1346, 1302, 1106, 1027cm⁻¹; ¹H NMR (CDCl₃, 300 MHz) δ 7.32-7.23 (m, 5H), 3.81-3.74 (m, 1H),3.71-3.55 (m, 5H), 3.47 (s, 2H), 3.41-3.19 (m, 9H), 2.46-2.11 (m, 5H),2.18 (s, 3H), 2.03-0.85 (series of multiplets, 20H), 0.93 (s, 3H), 0.93(d, J=6.35 Hz, 3H,), 0.67 (s, 3H); ¹³C NMR (CDCl₃, 75 MHz) δ 139.54,129.26, 128.32, 126.97, 81.26, 77.12, 75.17, 67.98, 67.42, 67.00, 62.50,58.41, 51.61, 51.54, 51.29, 46.66, 46.28, 42.46, 42.32, 39.72, 36.68,35.76, 35.16, 33.75, 32.38, 30.15, 28.59, 27.85, 27.19, 24.77, 24.15,23.53, 23.28, 23.22, 18.28, 12.60; HRFAB-MS (thioglycerol+Na⁺ matrix)m/e: ([M+H]⁺) 705.4929 (100%), calcd. 705.4928.

Compound 8: A suspension of 40 (0.109 g, 0.155 mmol) and LiAlH₄ (23.5mg, 0.62 mmol) in THF (20 mL) was stirred under N₂ overnight.Na₂SO₄.10H₂O was carefully added and stirred until no grey colorpersisted. Anhydrous Na₂SO₄ was added and the white precipitate wasfiltered out and rinsed with dry THF. After removal of solvent, theresidue was dissolved in minimum CH₂Cl₂ and filtered. The desiredproduct (0.091 g, 94% yield) was obtained as a colorless oil after thesolvent was removed. IR (neat) 3371, 3290, 3027, 2938, 2862, 2785, 1586,1493, 1453, 1377, 1347, 1098 cm⁻¹; ¹H NMR (CDCl₃, 300 MHz) δ 7.31-7.21(m, 5H), 3.65-3.53 (m, 4H), 3.47 (s, 2H), 3.42-3.34 (m, 2H), 3.30 (bs,1H), 3.26-3.20 (m, 1H), 3.14-3.09 (m, 1H), 2.89-2.81 (m, 6H), 2.39-2.27(m, 3H), 2.17 (s, 3H), 2.15-0.88 (series of multiplets, 29H), 0.93 (d,J=6.59 Hz, 3H), 0.92 (s, 3H), 0.67 (s, 3H); ¹³C NMR (CDCl₃, 75 MHz) δ139.34, 129.16, 128.24, 126.90, 80.75, 76.44, 74.29, 70.58, 69.88,69.75, 62.47, 58.27, 46.66, 46.47, 42.75, 42.63, 42.51, 42.35, 39.77,36.87, 35.73, 35.04, 33.77, 32.90, 30.38, 28.71, 27.70, 27.32, 24.89,24.09, 23.53, 23.36, 23.25, 18.24, 12.62; HRFAB-MS (thioglycerol+Na⁺matrix) m/e: ([M+H]⁺) 627.5199 (23.3%), calcd. 627.5213.

Compound CSA-7: To a solution of 23 (0.18 g, 0.28 mmol) in dry DMF (4mL) were added NaH (0.224 g, 60% in mineral oil, 5.60 mmol) and 1-bromooctane (0.48 mL, 2.80 mmol). The suspension was stirred under N₂ at 65°C. overnight followed by the introduction of H₂O (60 mL) and extractionwith ether (4×20 mL). The combined extracts were washed with brine anddried over Na₂SO₄. SiO₂ chromatography (hexanes and 5% EtOAc in hexanes)afforded the desired product (0.169 g, 80% yield) as a pale yellowishoil. IR (neat) 2927, 2865, 2099, 1478, 1462, 1451, 1350, 1264, 1105cm⁻¹; ¹H NMR (CDCl₃, 300 MHz) δ 3.69-3.35 (series of multiplets, 15H),3.26-3.02 (series of multiplets, 4H), 2.19-2.02 (m, 3H), 1.97-1.16(series of multiplets, 37H), 1.12-0.99 (m, 2H), 0.92-0.86 (m, 9H), 0.65(s, 3H); ¹³C NMR (CDCl₃, 75 MHz) δ 80.69, 79.84, 76.13, 71.57, 71.15,65.07, 64.49, 64.39, 49.08, 48.99, 48.80, 46.68, 46.45, 42.72, 42.05,39.88, 35.74, 35.49, 35.36, 35.14, 32.42, 32.03, 30.01, 29.85, 29.81,29.76, 29.67, 29.48, 29.14, 27.92, 27.80, 27.70, 26.58, 26.42, 23.59,23.09, 22.92, 22.86, 18.11, 14.31, 12.65; HRFAB-MS (thioglycerol+Na⁺matrix) m/e: ([M+Na]⁺) 778.5685 (22.1%), calcd. 778.5683. The triazide(0.169 g, 0.224 mmol) and LiAlH₄ (0.025 g, 0.67 mmol) were suspended inanhydrous THF (10 mL) and stirred under N₂ at room temperature overnightfollowed by careful introduction of Na₂SO₄ hydrate. After the grey colordisappeared, anhydrous Na₂SO₄ was added and stirred. The whiteprecipitate was removed by filtration and washed with THF. After removalof solvent, the residue was dissolved in 1 M hydrochloric acid and theaqueous solution was extracted with ether (5 mL) once. The aqueoussolution was then made basic by adding 20% aqueous NaOH solutionfollowed by extraction with Et₂O (4×5 mL). The combined extracts werewashed, dried and concentrated. The residue was then subject to SiO₂chromatography (MeOH/CH₂Cl₂ (1:1) followed by MeOH/CH₂Cl₂/NH₃. H₂O(4:4:1)) to afford the desired product (0.091 g, 60% yield) as acolorless oil. IR (neat) 3361, 2927, 2855, 1576, 1465, 1351, 1105 cm⁻¹;¹H NMR (CD₃OD, 300 MHz) δ 4.86 (bs, 6H), 3.77-3.72 (m, 1H), 3.70-3.61(m, 1H), 3.57-3.53 (m, 3H), 3.43-3.38 (m, 4H), 3.34-3.27 (m, 2H),3.18-3.10 (m, 2H), 2.84-2.71 (m, 6H), 2.22-2.07 (m, 3H), 2.00-1.02(series of multiplets, 39H), 0.97-0.88 (m, 9H), 0.71 (s, 3H); ¹³C NMR(CD₃OD, 75 MHz) δ 82.20, 81.00, 77.62, 72.52, 72.06, 68.00, 67.92,67.39, 48.20, 47.53, 44.26, 43.40, 41.42, 41.15, 40.84, 40.35, 36.88,36.73, 36.42, 36.11, 34.24, 34.05, 33.94, 33.67, 33.17, 30.95, 30.72,30.62, 29.81, 29.35, 28.87, 28.79, 27.51, 24.57, 23.90, 23.83, 23.44,18.76, 14.62, 13.07; HRFAB-MS (thioglycerol matrix) m/e: ([M+H]⁺)678.6133 (100%), calcd. 678.6149.

Compound CSA-8: A suspension of 23 (0.126 g, 0.196 mmol) and LiAlH₄(0.037 g, 0.98 mmol) in THF (40 mL) was stirred at room temperatureunder N₂ overnight followed by careful addition of Na₂SO₄.10H₂O. Afterthe grey color in the suspension disappeared, anhydrous Na₂SO₄ was addedand stirred until organic layer became clear. The white precipitate wasremoved by filtration and washed with twice THF. The THF was removed invacuo, and the residue was subject to SiO₂ chromatography(MeOH/CH₂Cl₂/NH₃/H₂O (4:4:1)) to afford the desired product (0.066 g,60% yield) as a colorless oil. IR (neat) 3365, 2933, 2865, 1651, 1471,1455, 1339, 1103 cm⁻¹; ¹H NMR (CDCl₃/30% CD₃OD, 300 MHz) δ 4.43 (bs,7H), 3.74-3.68 (m, 1 II), 3.66-3.60 (m, 1H), 3.57-3.50 (m, 5H),3.34-3.25 (M, 2H), 3.17-3.06 (M, 2H), 2.84-2.74 (M, 6H), 2.19-2.01 (M,3H), 1.97-0.96 (series of multiplets, 27H), 0.94 (d, J=7.2 Hz, 3H), 0.92(s, 3H), 0.69 (s, 3H); ¹³C NMR (CDCl₃, 75 MHz) δ 80.44, 79.27, 75.77,66.59, 66.53, 65.86, 62.51, 46.21, 45.84, 42.55, 41.53, 40.09, 39.43,39.31, 39.02, 35.16, 34.93, 34.86, 34.57, 32.93, 32.71, 31.57, 28.66,28.33, 27.64, 27.22, 23.04, 22.40, 22.29, 17.60, 11.98; HRFAB-MS(thioglycerol+Na⁺ matrix) m/e: ([M+H]⁺) 566.4889 (8.9%), calcd.566.4897.

Example 5

This example includes a description of one or more exemplary synthesticprocedures for obtaining Compounds CSA-11 and 43-47.

Compound 43: Precursor compound 41 was prepared following the methodreported by D. H. R. Barton, J. Wozniak, S. Z. Zard, Tetrahedron, 1989,vol. 45, 3741-3754. A mixture of 41 (1.00 g, 2.10 mmol), ethylene glycol(3.52 mL, 63 mmol) and p-TsOH (20 mg, 0.105 mmol) was refluxed inbenzene under N₂ for 16 hours. Water formed during the reaction wasremoved by a Dean-Stark moisture trap. The cooled mixture was washedwith NaHCO₃ solution (50 mL) and extracted with Et₂O (50 mL, 2×30 mL).The combined extracts were washed with brine and dried over anhydrousNa₂SO₄. Removal of the solvent gave the product (1.09 g, 100%) as awhite glass. IR (neat) 2939, 2876, 1735, 1447, 1377, 1247, 1074, 1057,1039 cm⁻¹; ¹H NMR (CDCl₃; 300 MHz) δ 5.10 (t, J=2.70 Hz, 1H), 4.92 (d,J=2.69 Hz, 1H), 4.63-4.52 (m, 1H), 3.98-3.80 (m, 4H), 2.32 (t, J=9.51Hz, 1H), 2.13 (s, 3H), 2.08 (s, 3H), 2.05 (s, 3H), 2.00-1.40 (series ofmultiplets, 15H), 1.34-0.98 (m, 3H), 1.20 (s, 3H), 0.92 (s, 3H), 0.82(s, 3H); ¹³C NMR (CDCl₃, 75 MHz) δ 170.69, 170.63, 170.47, 111.38,75.07, 74.23, 70.85, 64.95, 63.43, 49.85, 44.73, 43.39, 41.11, 37.37,34.84, 34.80, 34.52, 31.42, 29.18, 27.02, 25.41, 24.16, 22.72, 22.57,22.44, 21.73, 21.63, 13.40; HRFAB-MS (thioglycerol+Na⁺ matrix) m/e:([M+H]) 521.3106 (38.6%), calcd. 521.3114. The triacetate (1.09 g, 2.10mmol) was dissolved in MeOH (50 mL). NaOH (0.84 g, 21 mmol) was added tothe solution. The suspension was then refluxed under N₂ for 24 hours.MeOH was then removed in vacuo and the residue was dissolved in Et₂O(100 mL) and washed with H₂O, brine, and then dried over anhydrousNa₂SO₄. The desired product (0.80 g, 96% yield) was obtained as whitesolid after removal of solvent. m.p. 199-200° C. IR (neat) 3396, 2932,1462, 1446, 1371, 1265, 1078, 1055 cm⁻¹; ¹H NMR (10% CD₃OD in CDCl₃, 300MHz) δ 4.08-3.83 (series of multiplets, 9H), 3.44-3.34 (m, 1H), 2.41 (t,J=9.28 Hz, 1H), 2.22-2.10 (m, 2H), 1.96-1.50 (series of multiplets,12H), 1.45-0.96 (series of multiplets, 4H), 1.32 (s, 3H), 0.89 (s, 3H),0.78 (s, 3H); ¹³C NMR (10% CD₃OD in CDCl₃, 75 MHz) δ 112.11, 72.35,71.57, 68.09, 64.54, 63.24, 49.36, 45.90, 41.48, 41.45, 39.18, 38.79,35.29, 34.71, 34.45, 29.90, 27.26, 26.60, 23.65, 22.54, 22.44, 22.35,13.46; HRFAB-MS (thioglycerol+Na⁺ matrix) m/e: ([M+Na]⁺) 417.2622(87.3%), calcd. 417.2617.

Compound 44: To a round-bottom flask were added 43 (0.80 g, 2.03 mmol)and dry THF (100 mL) followed by the addition of NaH (60% in mineraloil, 0.81 g, 20.3 mmol). The suspension was refluxed under N₂ for 30minutes before the addition of allyl bromide (1.75 mL, 20.3 mmol). After48 hours of reflux, another 10 eq. of NaH and allyl bromide were added.After another 48 hours, TLC showed no intermediates left. Cold water (50mL) was added to the cooled suspension. The resulted mixture wasextracted with Et₂O (60 mL, 2×30 mL). The combined extracts were washedwith brine and dried over anhydrous Na₂SO₄. SiO₂ column chromatography(6% EtOAc in hexanes) gave the desired product (0.94 g, 90% yield) as apale yellow oil. IR (neat) 3076, 2933, 2866, 1645, 1446, 1423, 1408,1368, 1289, 1252, 1226, 1206, 1130, 1080, 1057 cm⁻¹; ¹H NMR (CDCl₃, 300MHz) δ 6.02-5.84 (m, 3H), 5.31-5.04 (m, 6H), 4.12-4.05 (m, 2H),4.01-3.81 (m, 7H), 3.70 (dd, J=12.94, 5.62 Hz, 1H), 3.55 (t, J=2.56 Hz,1H), 3.33 (d, J=2.93 Hz, 1H), 3.18-3.08 (m, 1H), 2.65 (t, J=10.01 Hz,1H), 2.32-2.14 (m, 3H), 1.84-1.45 (series of multiplets, 10H), 1.41-1.22(m, 3H), 1.27 (s, 3H), 1.14-0.92 (m, 2H), 0.89 (s, 3H), 0.75 (s, 3H);¹³C NMR (CDCl₃, 75 MHz) δ 136.38, 136.07, 136.00, 116.31, 115.54,115.38, 112.34, 80.07, 79.22, 75.05, 69.83, 69.34, 68.82, 65.14, 63.24,48.80, 45.96, 42.47, 42.15, 39.40, 35.55, 35.16, 35.15, 29.04, 28.22,27.52, 24.21, 23.38, 23.11, 22.95, 22.58, 13.79; HRFAB-MS(thioglycerol+Na⁺ matrix) m/e: ([M+Na]⁺) 537.3549 (100%), calcd.537.3556.

Compound 45: To the solution of 44 (0.94 g, 1.83 mmol) in dry THF (50mL) was added 9-BBN (0.5 M solution in THF, 14.7 mL, 7.34 mmol) and themixture was stirred under N₂ at room temperature for 12 hours before theaddition of 20% NaOH solution (4 mL) and 30% H₂ 02 solution (4 mL). Theresulted mixture was then refluxed for an hour followed by the additionof brine (100 mL) and extracted with EtOAc (4×30 mL). The combinedextracts were dried over anhydrous Na₂SO₄. After the removal of solvent,the residue was purified by SiO₂ column chromatography (EtOAc followedby 10% MeOH in CH₂Cl₂) to give the product (0.559 g, 54% yield) as acolorless oil. IR (neat) 3410, 2933, 2872, 1471, 1446, 1367, 1252, 1086cm⁻¹; ¹H NMR (CDCl₃, 300 MHz) δ 4.02-3.52 (series of multiplets, 17H),3.41-3.35 (m, 1H), 3.29 (d, J=2.44 Hz, 1H), 3.22-3.15 (m, 3H), 2.58 (t,J=10.01 Hz, 1H), 2.27-1.95 (m, 3H), 1.83-1.48 (series of multiplets,16H), 1.40-0.93 (series of multiplets, 5H), 1.27 (s, 3H), 0.90 (s, 3H),0.75 (s, 3H); ¹³C NMR (CDCl₃, 75 MHz) δ 112.41, 80.09, 79.09, 76.31,66.70, 66.02, 65.93, 64.80, 63.26, 61.53, 61.25, 60.86, 48.59, 45.80,42.51, 41.72, 39.10, 35.36, 35.02, 34.98, 32.87, 32.52, 32.40, 28.88,27.94, 27.21, 24.33, 23.02, 22.84 (2 C's), 22.44, 13.69; HRFAB-MS(thioglycerol+Na⁺ matrix) m/e: ([M+Na]⁺) 591.3881 (100%), calcd.591.3873.

Compound 46: To a solution of 45 (0.559 g, 0.98 mmol) in acetone (40 mL)and water (4 mL) was added PPTS (0.124 g, 0.49 mmol) and the solutionwas refluxed under N₂ for 16 hours. The solvent was removed underreduced pressure. Water (40 mL) was then added to the residue and themixture was extracted with EtOAc (40 mL, 2×20 mL). The combined extractswere washed with brine, dried and evaporated to dryness. SiO₂ columnchromatography (8% MeOH in CH₂C12) of the residue afforded the desiredproduct (0.509 g, 98% yield) as clear oil. IR (neat) 3382, 2941, 2876,1699, 1449, 1366, 1099 cm⁻¹; ¹H NMR (CDCl₃, 300 MHz) δ 3.83-3.72 (m,8H), 3.66 (t, J=5.62 Hz, 2H), 3.54 (bs, 2H), 3.43-3.28 (m, 4H),3.24-3.12 (m, 2H), 2.26-2.00 (m, 4H), 2.08 (s, 3H), 1.98-1.50 (series ofmultiplets, 15H), 1.42-0.96 (series of multiplets, 6H), 0.90 (s, 3H),0.62 (s, 3H); 13C NMR (CDCl₃, 75 MHz) δ 210.49, 78.87 (2 C's), 76.30,66.86, 66.18, 65.69, 61.74, 61.43, 60.71, 55.31, 48.05, 43.02, 41.58,39.53, 35.28, 35.09, 34.96, 32.77, 32.70, 32.31, 31.12, 28.72, 27.88,27.14, 23.47, 22.75, 22.47, 22.34, 13.86; HRFAB-MS (thioglycerol+Na⁺matrix) m/e: ([M+Na]⁺) 547.3624 (100%), calcd. 547.3611.

Compound 47: To a solution of 46 (0.18 g, 0.344 mmol) in dry CH₂Cl₂ (10mL) at 0° C. was added Et₃ N (0.168 mL, 1.20 mmol) followed by theaddition of mesyl chloride (0.088 mL, 1.13 mmol). After 10 minutes, H₂O(3 mL) and brine (30 mL) were added. The mixture was extracted withEtOAc (30 mL, 2×10 mL) and the extracts were washed with brine and driedover anhydrous Na₂SO₄. After removal of solvent, the residue wasdissolved in DMSO (5 mL) and NaN₃ (0.233 g, 3.44 mmol). The suspensionwas heated up to 50° C. under N₂ for 12 hours. H₂O (50 mL) was added tothe cool suspension and the mixture was extracted with EtOAc (30 mL,2×10 mL) and the extracts were washed with brine and dried overanhydrous Na₂SO₄. SiO₂ column chromatography (EtOAc/hexanes 1:5)afforded the product (0.191 g, 88% yield for two steps) as a pale yellowoil. IR (neat) 2933, 2872, 2096, 1702, 1451, 1363, 1263, 1102 cm⁻¹; ¹HNMR (CDCl₃, 300 MHz) δ 3.72-3.64 (m, 2H), 3.55-3.24 (series ofmultiplets, 11H), 3.18-3.02 (m, 2H), 2.22-2.02 (m, 4H), 2.08 (s, 3H),1.95-1.46 (series of multiplets, 15H), 1.38-0.96 (series of multiplets,6H), 0.89 (s, 3H), 0.62 (s, 3H); ¹³C NMR (CDCl₃, 75 MHz) δ 210.36,79.69, 79.22, 75.98, 65.08, 64.80, 64.53, 55.31, 48.93, 48.86, 48.76,48.06, 43.03, 41.91, 39.66, 35.44, 35.31, 35.12, 31.04, 29.77, 29.69,29.67, 28.99, 28.10, 27.65, 23.60, 22.99, 22.95, 22.50, 14.00; HRFAB-MS(thioglycerol+Na⁺ matrix) m/e: ([M+Na]⁺) 622.3820 (100%), calcd.622.3805.

Compound CSA-11: Compound 47 (0.191 g, 0.319 mmol) was dissolved in dryTHF (20 mL) followed by the addition of LiAlH₄ (60.4 mg, 1.59 mmol). Thegrey suspension was stirred under N₂ at room temperature for 12 hours.Na₂SO₄.10H₂O powder was carefully added. After the grey color in thesuspension disappeared, anhydrous Na₂SO₄ was added and the precipitatewas filtered out. After the removal of solvent, the residue was purifiedby column chromatography (silica gel, MeOH/CH₂Cl₂/28% NH₃.H₂O 3:3:1).After most of the solvent was rotavapped off from the fractionscollected, 5% HCl solution (2 mL) was added to dissolve the milkyresidue. The resulted clear solution was then extracted with Et₂O (2×10mL). 20% NaOH solution was then added until the solution became stronglybasic. CH₂Cl₂ (20 mL, 2×10 mL) was used to extract the basic solution.The combined extracts were dried over anhydrous Na₂SO₄ and removal ofsolvent gave the desired product (0.115 g, 69% yield) as a colorlessoil. From ¹H NMR it appears that this compound was a mixture of twostereoisomers at C₂₀ with a ratio of approximately 9:1. Thestereoisomers were not separated, but used as recovered. Spectra for themost abundant isomer: IR (neat) 3353, 2926, 2858, 1574, 1470, 1366, 1102cm⁻¹; ¹H NMR (20% CDCl₃ in CD₃OD, 300 MHz) δ 4.69 (bs, 7H), 3.76-3.69(m, 1H), 3.63-3.53 (m, 5H), 3.50-3.40 (m, 1H), 3.29 (bs, 1H), 3.18-3.07(m, 2H), 2.94-2.83 (m, 1H), 2.81-2.66 (m, 5H), 2.23-2.06 (m, 4H),1.87-1.50 (series of multiplets, 15H), 1.39-0.96 (series of multiplets,6H), 1.11 (d, J=6.10 Hz, 3H), 0.93 (s, 3H), 0.75 (s, 3H); ¹³C NMR (20%CDCl₃ in CD₃OD, 75 MHz) δ 81.46, 80.67, 77.32, 70.68, 67.90, 67.66,67.18, 50.32, 47.17, 43.30, 43.06, 40.74, 40.64, 40.38, 40.26, 36.31,36.28, 35.93, 34.30, 34.02, 33.29, 29.63, 29.31, 28.43, 26.10, 24.67,24.09, 23.96, 23.50, 13.30 for the major isomer; HRFAB-MS(thioglycerol+Na⁺ matrix) m/e: ([M+H]⁺) 524.4431 (64.2%), calcd.524.4427.

Example 6

This example includes a description of one or more exemplary synthesticprocedures for obtaining Compounds CSA-10 and 48-49.

Compound 48: To a solution of 23 (0.15 g, 0.233 mmol) in dry CH₂Cl₂ (15mL) at 0° C. was added Et₃ N (48.8 μL, 0.35 mmol) followed by theaddition of CH₃SO₂C1 (21.7 μL, 0.28 mmol). The mixture was stirred for15 minutes before H₂O (3 mL) was added. Saturated NaCl solution (20 mL)was then added, and the mixture was extracted with EtOAc (40 mL, 2×20mL). The combined extracts were washed with brine and dried overanhydrous Na₂SO₄. The solvent was rotovapped off and to the residue wereadded NaBr (0.12 g, 1.17 mmol) and DMF (10 mL). The suspension washeated up to 80° C. under N₂ for 2 hours. DMF was removed under vacuumand the residue was chromatographed on silica (EtOAc/hexanes 1:10) togive the desired product (0.191 g, 97% yield) as a pale yellow oil. ¹HNMR (CDCl₃, 300 MHz) δ 3.69-3.35 (series of multiplets, 13H), 3.28-3.02(series of multiplets, 4H), 2.18-2.04 (m, 3H), 2.00-1.60 (series ofmultiplets, 16H), 1.58-0.96 (series of multiplets, 11H), 0.92 (d, J=6.34Hz, 3H), 0.89 (s, 3H), 0.66 (s, 3H); ¹³C NMR (CDCl₃, 75 MHz) δ 80.62,79.81, 76.08, 65.07, 64.50, 64.34, 49.03, 48.98, 48.79, 46.49, 46.46,42.73, 42.02, 39.85, 35.47, 35.34, 35.12, 34.79, 34.72, 29.82, 29.80,29.74, 29.11, 27.91, 27.78, 27.69, 23.55, 23.07, 22.88, 18.10, 12.62;HRFAB-MS (thioglycerol+Na⁺ matrix) m/e: ([M−H]⁺) 706.3609 (63.1%),calcd. 706.3591; 704.3616 (52.8%), calcd. 704.3611.

Compound 49: Compound 48 (0.191 g, 0.269 mmol) and 23 (0.295 g, 0.459mmol) was dissolved in DMF (3 mL, distilled over BaO at 6 mm Hg beforeuse) followed by the addition of NaH (0.054 g, 60% in mineral oil). Thesuspension was stirred under N₂ at room temperature for 24 hours. H₂O(100 mL) was added to quench excess NaH and the mixture was thenextracted with Et₂O (40 mL, 3×20 mL) and the combined extracts werewashed with brine and dried over anhydrous Na₂SO₄. The desired product(0.177 g, 52% yield based on compound 23) was obtained as a pale yellowoil after SiO₂ chromatography (EtOAc/hexanes 1:6, then 1:2). IR (neat)2940, 2862, 2095, 1472, 1456, 1362, 1263, 1113 cm⁻¹; ¹H NMR (CDCl₃, 300MHz) δ 3.68-3.35 (series of multiplets, 26H), 3.28-3.02 (series ofmultiplets, 8H), 2.20-2.04 (m, 6H), 1.96-1.60 (series of multiplets,30H), 1.52-0.98 (series of multiplets, 12H), 0.91 (d, J=6.59 Hz, 6H),0.89 (s, 6H), 0.65 (s, 6H); ¹³C NMR (CDCl₃, 75 MHz) δ 80.68, 79.83,76.13, 71.71, 65.06, 64.48, 64.39, 49.08, 48.98, 48.80, 46.64, 46.44,42.71, 42.04, 39.88, 35.73, 35.49, 35.36, 35.14, 32.41, 29.84, 29.81,29.76, 29.14, 27.92, 27.78, 27.69, 26.58, 23.59, 23.08, 22.92, 18.12,12.64.

Compound CSA-10: Compound 49 (0.219 g, 0.173 mmol) was dissolved in dryTHF (10 mL) followed by the addition of LiAlH₄ (65 mg, 1.73 mmol). Thegrey suspension was stirred under N₂ at room temperature for 12 hours.Na₂SO₄.10H₂O powder was carefully added. After the grey color in thesuspension disappeared, anhydrous Na₂SO₄ was added and the precipitatewas filtered out. After the removal of solvent, the residue was purifiedby column chromatography (silica gel, MeOH/CH₂Cl₂/28% NH₃.H₂O2.5:2.5:1). After most of the solvent was rotavapped off from thefractions collected, 5% HCl solution (2 mL) was added to dissolve themilky residue. The resulted clear solution was then extracted with Et₂O(2×10 mL). 20% NaOH solution was then added until the solution becamestrongly basic. CH₂Cl₂ (20 mL, 2×10 mL) was used to extract the basicsolution. The combined extracts were dried over anhydrous Na₂SO₄ andremoval of solvent gave the desired product (0.147 g, 76% yield) as awhite glass. IR (neat) 3364, 3287, 2934, 2861, 1596, 1464, 1363, 1105cm⁻¹; ¹H NMR (20% CDCl₃ in CD₃OD, 500 MHz) δ 4.74 (bs, 12H), 3.75-3.70(m, 2H), 3.65-3.61 (m, 2H), 3.57-3.52 (m, 6H), 3.40 (t, J=3.60 Hz, 4H),3.30 (bs, 4H), 3.16-3.10 (m, 4H), 2.84-2.73 (m, 12H), 2.18-2.07 (m, 6H),1.97-1.61 (series of multiplets, 30H), 1.58-0.98 (series of multiplets,24H), 0.95 (d, J=6.84 Hz, 6H), 0.94 (s, 6H), 0.70 (s, 6H); ¹³C NMR (20%CDCl₃ in CD₃OD, 125 MHz) δ 81.70, 80.52, 77.09, 72.34, 67.75 (2 C's),67.07, 47.80, 47.13, 43.76, 42.87, 41.20, 40.65, 40.58, 40.14, 36.43,36.25, 36.08, 35.77, 34.15, 33.87 (2 C's), 33.18, 29.55, 28.92, 28.47,28.42, 27.25, 24.27, 23.54, 23.41, 18.70, 13.07; HRFAB-MS(thioglycerol+Na⁺ matrix) m/e: ([M+H]⁺) 1113.9625 (68.8%), calcd.1113.9610.

Example 7

This example includes a description of one or more exemplary synthesticprocedures for obtaining Compounds 111-113 and 116a-d.

Compounds 116a-d: Representative procedure: preparation of 116b. NaH(0.06 g, 60% in mineral oil, 1.49 mmol) and propyl bromide (0.136 mL,1.49 mmol) were added to a DMF solution of compound 23 (described in Liet al., J. Am. Chem. Soc. 1998, 120, 2961) (0.096 g, 0.149 mmol). Thesuspension was stirred under N₂ for 24 hr. H₂O (20 mL) was added, andthe mixture was extracted with hexanes (3×10 mL). The combined extractswere dried over Na₂SO₄ and concentrated in vacuo. Silica gelchromatography (10% EtOAc in hexanes) afforded the desired product (92mg, 90% yield) as a pale yellow oil. ¹H NMR (CDCl₃, 500 MHz) δ 3.68-3.64(m, 1H), 3.61-3.57 (m, 1H), 3.52 (t, J=6.1 Hz, 2H), 3.49 (bs, 1H),3.46-3.35 (m, 10H), 3.25 (d, J=2.4 Hz, 1H), 3.23-3.19 (m, 1H), 3.16-3.11(m, 1H), 3.09-3.03 (m, 1H), 2.17-2.03 (m, 3H), 1.95-1.55 (m, 17H),1.51-1.40 (m, 4H), 1.38-1.17 (m, 5H), 1.11-0.96 (m, 3H), 0.93-0.89 (m,9H), 0.65 (s, 3H); ¹³C NMR (CDCl₃, 75 MHz) δ 80.64, 79.79, 76.08, 72.67,71.59, 65.01, 64.44, 64.33, 49.04, 48.94, 48.75, 46.61, 46.40, 42.68,42.00, 39.83, 35.72, 35.45, 35.30, 35.10, 32.38, 29.81, 29.77, 29.72,29.09, 27.88, 27.76, 27.65, 26.52, 23.55, 23.12, 23.04, 22.87, 18.06,12.60, 10.79; HRFAB-MS (thioglycerol+Na⁺ matrix) m/e: ([M+Na]⁺) 708.4910(23.5%), calcd. 708.4920.

Compounds 111, CSA-17, and 113: Representative procedure: preparation ofCSA-17. Compound 116b (0.092 g, 0.134 mmol) was dissolved in THF (10 mL)followed by the addition of LiAlH₄ (0.031 g, 0.81 mmol). The suspensionwas stirred under N₂ for 12 hr. Na₂SO₄O.10H₂O (about 1 g) was thencarefully added. After the gray color in the suspension dissipated,anhydrous Na₂SO₄ was added, and the precipitate was removed byfiltration. Concentration and silica gel chromatography (CH₂Cl₂/MeOH/28%NH₃.H₂O 12:6:1, then 10:5:1) yielded a glass which was dissolved in 1 MHCl (2 mL). The resulting clear solution was washed with Et₂O (2×10 mL).20% NaOH solution was added to the aqueous phase until the solutionbecame strongly basic. CH₂Cl₂ (3×10 mL) was used to extract the basicsolution. The combined extracts were dried over anhydrous Na₂SO₄ andconcentrated in vacuo to give the desired product (0.045 g, 55% yield)as a white glass. ¹H NMR (about 20% CDCl₃ in CD₃OD, 500 MHz) δ 4.73 (bs,6H), 3.74-3.70 (m, 1H), 3.65-3.61 (m, 1H), 3.55 (t, J=6.3 Hz, 2H),3.42-3.38 (m, 4H), 3.33-3.30 (m, 2H), 3.16-3.10 (m, 2H), 2.83-2.73 (m,6H), 2.18-2.06 (m, 3H), 1.96-1.20 (series of multiplets, 26H), 1.12-0.98(m, 3H), 0.95-0.92 (m, 9H), 0.70 (s, 3H); ¹³C NMR (about 20% CDCl₃ inCD₃OD, 75 MHz) δ 81.67, 80.49, 77.04, 73.44, 72.28, 67.77, 67.71, 67.06,47.74, 47.08, 43.75, 42.82, 41.21, 40.60, 40.56, 40.12, 36.47, 36.19,36.04, 35.74, 34.09, 33.82, 33.78, 33.16, 29.49, 28.87, 28.43, 27.18,24.22, 23.66, 23.49, 23.40, 18.64, 13.04, 11.03; HRFAB-MS(thioglycerol+Na⁺ matrix) m/e: ([M+H]⁺) 608.5348 (100%), calcd.608.5330. 111: ¹H NMR (about 20% CDCl₃ in CD₃OD, 500 MHz) δ 4.79 (bs,6H), 3.74-3.71 (m, 1H), 3.66-3.62 (m, 1H), 3.55 (t, J=6.1 Hz, 2H), 3.52(bs, 1H), 3.38-3.28 (series of multiplets, 4H), 3.33 (s, 3H), 3.16-3.10(m, 2H), 2.83-2.72 (m, 6H), 2.19-2.07 (m, 3H), 1.97-1.62 (series ofmultiplets, 15H), 1.58-1.20 (series of multiplets, 9H), 1.13-0.98 (m,3H), 0.95 (d, J=6.3 Hz, 3H), 0.93 (s, 3H), 0.70 (s, 3H); ¹³C NMR (about20% CDCl₃ in CD₃OD, 75 MHz) δ 81.82, 80.65, 77.20, 74.43, 67.85, 67.18,58.90, 47.80, 47.22, 43.91, 43.01, 41.31, 40.78, 40.69, 40.22, 36.63,36.35, 36.18, 35.86, 34.27, 33.97, 33.26, 29.60, 29.03, 28.58, 28.53,27.14, 24.33, 23.61, 23.45, 18.68, 13.06; HRFAB-MS (thioglycerol+Na⁺matrix) m/e: ([M+Na]⁺) 602.4855 (100%), calcd. 602.4873. 113: ¹H NMR(about 50% CDCl₃ in CD₃OD, 500 MHz) 4.08 (bs, 6H), 3.71-3.67 (m, 1H),3.62-3.58 (m, 1H), 3.53 (t, J=6.3 Hz, 2H), 3.49 (bs, 1H), 3.43-3.38 (m,4H), 3.31-3.27 (m, 2H), 3.14-3.07 (m, 2H), 2.83-2.73 (m, 6H), 2.16-2.03(m, 3H), 1.93-1.17 (series of multiplets, 30H), 1.10-0.96 (m, 3H),0.93-0.89 (m, 9H), 0.67 (s, 3H); ¹³C NMR (about 50% CDCl₃ in CD₃OD, 75MHz) δ 80.51, 79.35, 75.85, 71.29, 70.83, 66.73, 66.62, 65.96, 46.68,45.98, 42.59, 41.63, 40.20, 39.53, 39.43, 39.21, 35.34, 35.04, 35.00,34.71, 33.11, 32.90, 32.82, 32.00, 29.15, 28.49, 28.15, 27.75, 27.35,26.22, 23.18, 22.60, 22.45, 22.34, 17.77, 13.75, 12.22; HRFAB-MS(thioglycerol+Na⁺ matrix) m/e: ([M+H]⁺) 636.5679 (100%), calcd.636.5669.

Example 8

This example includes a description of one or more exemplary synthesticprocedures for obtaining Compounds 106 and 124.

Compound 124: Compound 47 (0.256 g, 0.489 mmol) was dissolved in CH₂Cl₂(10 mL), and cooled to 0° C. followed by the addition of Na₂HPO₄ (0.69g, 4.89 mmol) and urea-hydrogen peroxide complex (UHP) (0.069 g, 0.733mmol). Trifluoroacetic anhydride (TFAA) (0.138 mL, 0.977 mmol) was thenadded dropwise. The suspension was stirred for 12 hr, and additional UHP(23 mg, 0.25 mmol) and TFAA (0.069 mL, 0.49 mmol) were added. Afteranother 12 hr, H₂O (30 mL) was added, and the resulting mixture wasextracted with EtOAc (3×20 mL). The combined extracts were washed withbrine (50 mL), dried over anhydrous Na₂SO₄, and concentrated in vacuo.SiO₂ chromatography (EtOAc/hexanes 1:5) afforded the desired product(0.145 g, 55% yield) as a colorless oil. ¹H NMR (CDCl₃, 300 MHz) δ 5.21(dd, J=9.3 and 7.3 Hz, 1H), 3.70-3.57 (m, 2H), 3.55 (t, J=6.0 Hz, 2H),3.43-3.37 (m, 6H), 3.32-3.25 (m, 3H), 3.17-3.02 (m, 2H), 2.28-2.05 (m,4H), 2.03 (s, 3H), 1.86-1.19 (series of multiplets, 19H), 0.97 (dd,J=14.5 and 3.3 Hz, 1H), 0.90 (s, 3H), 0.78 (s, 3H); ¹³C NMR (CDCl₃, 75MHz) δ 171.08, 79.71, 78.03, 75.72, 75.53, 65.41, 65.04, 64.53, 48.79,48.70, 46.49, 41.92, 39.44, 37.81, 35.45, 35.22, 35.10, 29.73, 29.63,28.89, 28.33, 27.50, 27.34, 23.39, 22.97, 22.92, 21.28, 12.72; HRFAB-MS(thioglycerol+Na⁺ matrix) m/e: ([M−H]⁺) 614.3798 (24.5%), calcd.614.3778.

Compound 106: Compound 124 (0.145 g, 0.236 mmol) was dissolved in CH₂Cl₂(2 mL) and MeOH (1 mL). 20% NaOH solution (0.2 mL) was added. Themixture was stirred for 12 hr, and anhydrous Na₂SO₄ was used to removewater. After concentration in vacuo, the residue was purified by silicagel chromatography (EtOAc/hexanes 1:3) to afford the desired product(0.124 g, 92% yield) as a colorless oil. ¹H NMR (CDCl₃, 300 MHz) δ 4.29(bs, 1H), 3.69-3.60 (m, 2H), 3.52 (t, J=6.0 Hz, 2H), 3.45-3.32 (m, 8H),3.26 (d, J=2.7 Hz, 1H), 3.17-3.02 (m, 2H), 2.19-1.94 (m, 4H), 1.90-1.62(series of multiplets, 13H), 1.57-1.20 (series of multiplets, 7H), 0.97(dd, J=14.3 and 3.1 Hz, 1H), 0.90 (s, 3H), 0.73 (s, 3H); ³C NMR (CDCl₃,75 MHz) δ 79.69, 78.03, 75.47, 73.38, 65.46, 65.00, 64.47, 48.87, 48.68,46.83, 41.93, 39.71, 37.87, 35.43, 35.20, 35.09, 29.96, 29.69, 29.59,29.53, 28.89, 28.44, 27.48, 23.72, 22.91, 22.71, 11.77. The alcohol(0.124 g, 0.216 mmol) was dissolved in dry THF (20 mL) followed by theaddition of LiAlH₄ (33 mg, 0.866 mmol). The gray suspension was stirredunder N₂ for 12 hr. Na₂SO₄O.10 H₂O (about 2 g) was carefully added.After the gray color in the suspension dissipated, anhydrous Na₂SO₄ wasadded and the precipitate was removed by filtration. After the removalof solvent, the residue was purified by column chromatography (SiO₂,MeOH/CH₂Cl₂/28% NH₃.H₂O 2.5:2.5:1). After concentration of the relevantfractions, 1 M HCl (2 mL) was added to dissolve the milky residue. Theresulting clear solution was washed with Et₂O (2×10 mL). To the aqueousphase, 20% NaOH solution was added until the solution became stronglybasic. CH₂Cl₂ (20 mL, 2×10 mL) was used to extract the basic solution.The combined extracts were dried over anhydrous Na₂SO₄ and removal ofsolvent gave the desired product (0.050 g, 47% yield) as a colorlessoil. ¹H NMR (20% CDCl₃ in CD₃OD, 300 MHz) δ 4.77 (s, 7H), 4.25 (t, J=8.5Hz, 1H), 3.75-3.68 (m, 1H), 3.66-3.58 (m, 1H), 3.55 (t, J=6.1 Hz, 2H),3.48-3.41 (m, 1H), 3.34 (bs, 1H), 3.30 (d, J=3.6 Hz, 1H), 3.17-3.08 (m,2H), 2.86-2.70 (m, 6H), 2.20-1.91 (m, 4H), 1.88-1.16 (series ofmultiplets, 19H), 1.00 (dd, J=14.2 and 3.0 Hz, 1H), 0.93 (s, 3H), 0.73(s, 3H); 13C NMR (20% CDCl₃ in CD₃OD, 75 MHz) δ 80.62, 79.12, 76.74,73.77, 68.50, 67.79, 67.17, 47.69, 43.04, 40.76, 40.64, 40.62, 40.22,39.01, 36.32, 36.25, 35.94, 34.27, 33.97, 33.72, 30.13, 29.53, 28.43,24.48, 23.58, 23.40, 12.38; HRFAB-MS (thioglycerol+Na⁺ matrix) m/e:([M+H]⁺) 496.4108 (100%), calcd. 496.4114.

Example 9

This example includes a description of one or more exemplary synthesticprocedures for obtaining Compounds 109 and 126-129.

Compound 126: Compound 125 (2.30 g, 3.52 mmol) was dissolved in MeOH (50mL) and CH₂Cl₂ (100 mL). A small amount of Et₃N was added, and thesolution was cooled to −78° C. Ozone was bubbled through the solutionuntil a blue color persisted. Me₂S (4 mL) was introduced followed by theaddition of NaBH₄ (0.266 g, 0.703 mmol) in MeOH (10 mL). The resultingsolution was allowed to warm and stir overnight. The solution wasconcentrated in vacuo, and brine (60 mL) was added. The mixture wasextracted with EtOAc (40 ml, 2×30 mL), and the combined extracts werewashed with brine and dried over anhydrous Na₂SO₄. Silica gelchromatography (EtOAc) afforded the product (1.24 g, 76% yield) as awhite solid. m.p. 219-220 C; ¹H NMR (CDCl₃, 300 MHz) δ 5.10 (t, J=2.8Hz, 1H), 4.90 (d, J=2.7 Hz, 1H), 3.73-3.59 (m, 2H), 3.56-3.44 (m, 1H),2.13 (s, 3H), 2.09 (s, 3H), 2.07-0.95 (series of multiplets, 23H), 0.91(s, 3H), 0.83 (d, J=6.3 Hz, 3H), 0.74 (s, 3H); ¹³C NMR (CDCl₃, 75 MHz) δ170.84, 170.82, 75.63, 71.77, 71.03, 60.73, 48.10, 45.26, 43.54, 41.16,38.78, 37.89, 35.00, 34.43, 32.26, 31.50, 30.60, 29.07, 27.50, 25.70,22.96, 22.71, 21.81, 21.63, 18.18, 12.35; HRFAB-MS (thioglycerol+Na⁺matrix) m/e: ([M+H]⁺) 465.3197 (20%), calcd. 465.3216.

Compound 127: Compound 126 (1.24 g, 2.67 mmol) was dissolved in MeOH (30mL), and NaOH (0.54 g, 13.4 mmol) was added. The suspension was refluxedunder N₂ for 24 hr. The MeOH was removed in vacuo followed by theaddition of H₂O (50 mL). The precipitate was filtered, washed with H₂Oand then dried in vacuo to give a white solid (1.02 g). This solid wasdissolved in DMF (40 mL) followed by the sequential addition of NEt₃(1.12 mL, 8.02 mmol), DMAP (16.3 mg, 0.13 mmol) and trityl chloride(1.49 g, 5.34 mmol). The suspension was stirred under N₂ for 12 hr andthen heated up to 50° C. for 24 hr. H₂O (100 mL) was added to the cooledsuspension, and the mixture was extracted with EtOAc (3×50 mL). Thecombined extracts were washed with brine (100 mL), dried over anhydrousNa₂SO₄, and concentrated in vacuo. Silica gel chromatography (EtOAc)afforded the product (1.20 g, 72% yield) as a pale yellow glass. To thisglass was added dry THF (80 mL) and NaH (60% in mineral oil, 0.77 g,19.3 mmol). The suspension was refluxed under N₂ for half an hour beforethe introduction of allylbromide (1.67 mL, 19.3 mmol). After 48 hr atreflux, another 10 eq. of NaH and allylbromide were introduced. Afteranother 48 hr, the reaction mixture was cooled and H₂O (100 mL) wasslowly added. The resulting mixture was extracted with hexanes (3×50mL), and the combined extracts were washed with brine (100 mL) and driedover anhydrous Na₂SO₄. Silica gel chromatography (5% EtOAc in hexanes)afforded the product (1.27 g, 64% yield for all three steps) as a clearglass. ¹H NMR (CDCl₃, 300 MHz) δ 7.46-7.43 (m, 6H), 7.29-7.16 (m, 9H),5.98-5.81 (m, 3H), 5.29-5.18 (m, 3H), 5.14-5.03 (m, 3H), 4.11-3.97 (m,4H), 3.75-3.67 (m, 2H), 3.49 (bs, 1H), 3.32-3.13 (d, J=2.4 Hz, 1H),3.20-3.13 (m, 2H), 3.00 (m, 1H), 2.33-2.12 (m, 3H), 2.03-0.92 (series ofmultiplets, 19H), 0.88 (s, 3H), 0.78 (d, J=6.6 Hz, 3H), 0.65 (s, 3H);¹³C NMR (CDCl₃, 75 MHz) δ 144.71, 136.08, 136.04, 135.94, 128.80,127.76, 126.86, 116.30, 115.57, 86.53, 80.77, 79.20, 74.96, 69.42,69.34, 68.81, 62.00, 46.87, 46.48, 42.67, 42.11, 39.90, 36.15, 35.50,35.14, 35.10, 33.23, 28.99, 28.09, 27.75, 27.56, 23.36, 23.32, 23.12,18.24, 12.66; HRFAB-MS (thioglycerol+Na⁺ matrix) m/e: ([M+Na]⁺) 765.4875(100%), calcd. 765.4859.

Compound 128: To a THF (40 mL) solution of 127 (1.27 g, 1.71 mmol) wasadded 9-BBN (0.5 M solution in THF, 17.1 mL). The mixture was stirredfor 12 hr before the addition of NaOH (20% solution, 10 mL) and H₂O₂(30% solution, 10 mL). The resulted mixture was refluxed for 1 hrfollowed by the addition of brine (100 mL) and extraction with EtOAc(4×30 mL). The combined extracts were dried over anhydrous Na₂SO₄ andconcentrated in vacuo. Silica gel chromatography (5% MeOH in CH₂Cl₂)afforded the product (1.26 g, 93% yield) as a clear glass. ¹H NMR (5%CD₃OD in CDCl₃, 300 MHz) O 7.46-7.43 (m, 6H), 7.32-7.20 (m, 9H), 3.94(s, 3H), 3.78-3.56 (m, 10H), 3.48 (bs, 1H), 3.32-3.26 (m, 2H), 3.24-3.12(m, 3H), 3.00 (dd, J=8.2 and 6.1 Hz, 1H), 2.23-1.96 (m, 3H), 1.90-0.95(series of multiplets, 25H), 0.90 (s, 3H), 0.77 (d, J=6.6 Hz, 3H), 0.66(s, 3H); ¹³C NMR (5% CD₃OD in CDCl₃, 75 MHz) δ 144.52, 128.64, 127.64,126.76, 86.43, 80.55, 79.31, 77.65, 77.23, 76.80, 76.06, 66.17, 66.01,65.41, 61.93, 61.20, 60.73, 60.39, 47.29, 46.08, 42.65, 41.62, 39.49,36.02, 35.10, 34.89, 34.77, 32.89, 32.71, 32.41, 32.26, 28.68, 27.70,27.51, 27.19, 23.26, 22.66, 22.50, 18.23, 12.34; HRFAB-MS(thioglycerol+Na⁺ matrix) m/e: ([M+Na]⁺) 819.5169 (100%), calcd.819.5099.

Compound 129: To a CH₂Cl₂ (50 mL) solution of compound 128 (1.26 g, 1.58mmol) at 0° C. was added Et₃N (0.92 mL, 6.60 mmol) followed by mesylchloride (0.47 mL, 6.05 mmol). After 15 minutes, H₂O (10 mL) wasfollowed by brine (80 mL). The mixture was extracted with EtOAc (60 mL,2×30 mL) and the combined extracts were dried over anhydrous Na₂SO₄.After removal of solvent in vacuo, the residue was dissolved in DMSO (10mL) and NaN₃ (1.192 g, 18.3 mmol) was added. The suspension was heatedto 60° C. under N₂ overnight. H₂O (100 mL) was added, and the mixturewas extracted with EtOAc (3×40 mL). The combined extracts were washedwith brine and dried over anhydrous Na₂SO₄. Removal of the solvent invacuo afforded a pale yellow oil. The oil was dissolved in MeOH (10 mL)and CH₂Cl₂ (20 mL) and TsOH (17.4 mg, 0.092 mmol) was added. After 12hr, saturated aqueous NaHCO₃ (20 mL) and brine (50 mL) were added andthe mixture was extracted with EtOAc (3×40 mL). The combined extractswere washed with brine (50 mL) and dried over anhydrous Na₂SO₄. Silicagel chromatography (EtOAc/hexanes 1:3) afforded the desired product(0.934, 94%) as a pale yellow oil. ¹H NMR (CDCl₃, 500 MHz) δ 3.75-3.70(m, 1H), 3.68-3.63 (m, 2H), 3.62-3.57 (m, 1H), 3.53 (t, J=6.1 Hz, 2H),3.50 (bs, 1H), 3.46-3.38 (m, 6H), 3.26 (d, J=2.4 Hz, 1H), 3.24-3.20 (m,1H), 3.16-3.12 (m, 1H), 3.10-3.04 (m, 1H), 2.17-2.04 (m, 3H), 1.96-1.63(m, 14H), 1.53-1.45 (m, 3H), 1.35-1.20 (m, 7H), 1.08-1.00 (m, 1H),0.97-0.88 (m, 1H), 0.94 (d, J=6.8 Hz, 3H), 0.89 (s, 3H), 0.67 (s, 3H);¹³C NMR (CDCl₃, 75 MHz) δ 80.64, 79.81, 76.06, 65.05, 64.49, 64.34,61.03, 49.02, 48.98, 48.78, 46.93, 46.53, 42.76, 42.01, 39.83, 39.14,35.46, 35.33, 35.12, 32.97, 29.79, 29.73, 29.10, 27.90, 27.68, 23.56,23.06, 22.88, 18.24, 12.60; HRFAB-MS (thioglycerol+Na⁺ matrix) m/e:([M+Na]⁺) 652.4285 (100%), calcd. 652.4295.

Compound 109: Compound 129 (0.245 g, 0.391 mmol) was dissolved in THF(30 mL) followed by the addition of LiAlH₄ (59 mg, 1.56 mmol). The graysuspension was stirred under N₂ 12 hr. Na₂SO₄.10H₂O powder (about 1 g)was carefully added. After the gray color in the suspension dissipated,anhydrous Na₂SO₄ was added and the precipitate was removed byfiltration. After the removal of solvent, the residue was purified bysilica gel chromatography (CH₂Cl₂/MeOH/28% NH₃.H₂O 10:5:1 then10:5:1.5). The solvent was removed from relevant fractions, and 1 M HCl(4 mL) was added to dissolve the residue. The resulting clear solutionwas extracted with Et₂O (3×10 mL). 20% NaOH solution was added until thesolution became strongly basic. CH₂Cl₂ (4×10 mL) was used to extract thebasic solution. The combined extracts were dried over anhydrous Na₂SO₄,and removal of solvent in vacuo gave the desired product (0.15 g, 71%yield) as a colorless oil. ¹H NMR (about 20% CD₃OD in CDCl₃, 500 MHz) δ4.73 (bs, 7H), 3.74-3.70 (m, 1H), 3.65-3.60 (m, 2H), 3.56-3.52 (m, 4H),3.31-3.28 (m, 2H), 3.16-3.09 (m, 2H), 2.82-2.71 (m, 6H), 2.19-2.06 (m,3H), 1.97-1.66 (series of multiplets, 15H), 1.58-1.48 (m, 3H), 1.38-0.98(m, 7H), 0.96 (d, J=6.8 Hz, 3H), 0.93 (s, 3H), 0.71 (s, 3H); ¹³C NMR(about 20% CD₃OD in CDCl₃, 75 MHz) δ 81.80, 80.60, 77.17, 67.88, 67.86,67.18, 60.73, 48.11, 47.28, 43.93, 42.99, 41.34, 40.76, 40.72, 40.24,39.70, 36.33, 36.18, 35.86, 34.29, 33.99, 33.96, 33.83, 29.60, 29.00,28.57, 28.54, 24.33, 23.59, 23.48, 18.86, 13.04; HRFAB-MS(thioglycerol+Na⁺ matrix) m/e: ([M+H]⁺) 552.4756 (100%), calcd.552.4772.

Example 10

This example includes a description of one or more exemplary synthesticprocedures for obtaining Compounds 108 and 130.

Compound 130: o-NO₂C6H₄SeCN (0.094 g, 0.21 mmol) and Bu₃P (0.095 mL,0.38 mmol) were stirred in dry THF (5 mL) at 0° C. for ½ hr followed bythe addition of compound 129 (0.10 g, 0.159 mmol) in THF (2 mL). Thesuspension was stirred for 1 hr followed by the addition of H₂O₂ (30%aqueous solution, 2 mL). The mixture was stirred for 12 hr followed byextraction with hexanes (4×10 mL). The combined extracts were dried overanhydrous Na₂SO₄. The desired product (0.035 g, 36% yield) was obtainedas pale yellowish oil after silical gel chromatography (10%EtOAc/hexanes). ¹H NMR (CDCl₃, 500 MHz) δ 5.73-5.66 (ddd, J=17.1, 10.2,8.3 Hz, 1H), 4.90 (dd, J=17.1, 2.0 Hz, 1H), 4.82 (dd, J=10.2 Hz, 1.96Hz, 1H), 3.68-3.64 (m, 1H), 3.62-3.58 (m, 1H), 3.54-3.26 (m, 9H),3.25-3.22 (m, 2H), 3.15-3.11 (m, 1H), 3.10-3.04 (m, 1H), 2.17-1.62(series of multiplets, 18H), 1.51-1.43 (m, 2H), 1.35-1.18 (m, 4H),1.06-0.91 (m, 2H), 1.02 (d, J=6.3 Hz, 3H), 0.90 (s, 3H), 0.68 (s, 3H);¹³C NMR (CDCl₃, 75 MHz) δ 145.50, 111.72, 80.60, 79.82, 76.09, 65.06,64.50, 64.45, 49.05, 48.97, 48.79, 46.43, 46.13, 42.76, 42.03, 41.30,39.84, 35.49, 35.34, 35.15, 29.82, 29.80, 29.75, 29.11, 28.00, 27.84,27.68, 23.56, 23.08, 22.95, 19.79, 12.87; HRFAB-MS (thioglycerol+Na⁺matrix) m/e: ([M+Na]⁺) 634.4167 (90.6%), calcd. 634.4169.

Compound 108: Compound 130 (0.105 g, 0.172 mmol) was dissolved in CH₂Cl₂(5 mL) and MeOH (5 mL) at −78° C. 03 was bubbled into the solution forca. 20 min. Me₂S (1 mL) was added followed, and the solvent was removedin vacuo. The residue was dissolved in THF (15 mL), and LiAlH₄ (0.033 g,0.86 mmol) was added. The suspension was stirred for 12 hr. Na₂SO₄.10H₂O(about 2 g) was carefully added. After the gray color of the suspensiondissipated, anhydrous Na₂SO₄ was added and the precipitate was removedby filtration. Concentration and silica gel chromatography(CH₂Cl₂/MeOH/28% NH₃.H₂O 10:5:1.5 then 9:6:1.8) yielded a white glass.To this material was added 1 M HCl (4 mL). The resulting clear solutionwas washed with Et₂O (3×10 mL). 20% NaOH solution was added to theaqueous phase until the solution became strongly basic. CH₂Cl₂ (4×10 mL)was used to extract the basic solution. The combined extracts were driedover anhydrous Na₂SO₄ and removal of solvent gave the desired product(0.063 g, 68% yield) as a colorless oil. ¹H NMR (about 10% CD₃OD inCDCl₃, 500 MHz) δ 4.76 (bs, 7H), 3.75-3.71 (m, 1H), 3.66-3.62 (m, 1H),3.58-3.52 (m, 4H), 3.33-3.29 (m, 2H), 3.22 (dd, J=10.5 and 7.6 Hz, 1H),3.15-3.09 (m, 2H), 2.81 (t, J=6.8 Hz, 2H), 2.76-2.71 (m, 4H), 2.19-2.08(m, 3H), 2.00-1.66 (series of multiplets, 14H), 1.58-1.45 (m, 3H),1.40-1.08 (m, 5H), 1.03 (d, J=6.8 Hz, 3H), 1.02-0.96 (m, 1H), 0.93 (s,3H), 0.72 (s, 3H); ¹³C NMR (about 10% CD₃OD in CDCl₃, 75 MHz) δ 81.74,80.64, 77.23, 67.95, 67.87, 67.18, 47.32, 44.59, 43.72, 43.01, 41.26,40.80, 40.71, 40.23, 40.02, 36.36, 36.20, 35.87, 34.27, 33.99, 33.90,29.60, 29.05, 28.58, 28.08, 24.49, 23.62, 23.46, 16.84, 13.12; HRFAB-MS(thioglycerol+Na⁺ matrix) m/e: ([M+H]) 538.4578 (4.7%), calcd. 538.4584.

Example 11

This example includes a description of one or more exemplary synthesticprocedures for obtaining Compounds CSA-21, 133-134 and CSA-15.

Compound CSA-21: Compound 115 (0.118 g, 0.183 mmol) was dissolved in dryCH₂Cl₂ (10 mL), and SO₃ pyridine complex (0.035 g, 0.22 mmol) was added.The suspension was stirred for 12 hr. The solvent was removed in vacuoto give white powder. To the white powder was added 1 M HCl (10 mL) andthe resulting mixture was extracted with CH₂Cl₂ (4×10 mL). The combinedextracts were dried over anhydrous Na₂SO₄. The desired product (0.11 g,84%) was obtained as a pale yellow oil after silica gel chromatography(10% MeOH in CH₂C2). ¹H NMR (about 10% CD₃OD in CDCl₃, 500 MHz) δ 4.03(t, J=6.8 Hz, 2H), 3.69-3.65 (m, 1H), 3.62-3.58 (m, 1H), 3.55 (t, J=6.1Hz, 2H), 3.51 (bs, 1H), 3.46-3.38 (m, 6H), 3.27 (d, J=2.4 Hz, 1H),3.26-3.21 (m, 1H), 3.18-3.07 (m, 2H), 2.18-2.03 (m, 3H), 1.95-1.47(series of multiplets, 19H), 1.40-0.96 (series of multiplets, 9H), 0.92(d, J=6.8 Hz, 3H), 0.91 (s, 3H), 0.66 (s, 3H); ¹³C NMR (about 10% CD₃ODin CDCl₃, 75 MHz) δ 80.43, 79.68, 75.87, 69.30, 64.82, 64.32, 64.14,48.78, 48.73, 48.50, 46.44, 46.21, 42.49, 41.76, 39.61, 35.36, 35.17,35.06, 34.85, 31.73, 29.53, 29.46, 29.44, 28.84, 27.68, 27.48, 27.38,25.91, 23.30, 22.75, 22.66, 17.70, 12.32; HRFAB-MS (thioglycerol+Na⁺matrix) m/e: ([M−H+2Na]⁺) 768.3831 (100%), calcd. 768.3843. The azideswere reduced by treating the triazide (0.11 g, 0.15 mmol) with Ph₃P(0.20 g, 0.77 mmol) in THF (10 mL) and H₂O (1 mL). The mixture wasstirred for 3 days. The solvent was removed in vacuo, and the residuewas purified by silica gel chromatography (CH₂Cl₂/MeOH/28% NH₃.H₂O12:6:1 then 10:5:1.5) to afford the desired product (0.077 g, 78% yield)as a glass. HCl in Et₂O (1 M, 0.5 mL) was added to the glass to give thecorresponding HCl salt. ¹H NMR (about 10% CDCl₃ in CD₃OD, 500 MHz) δ4.81 (s, 10H), 4.07-3.97 (m, 2H), 3.82 (bs, 1H), 3.71 (bs, 1H), 3.65 (t,J=5.2 Hz, 2H), 3.57 (bs, 1H), 3.37-3.30 (m, 2H), 3.22-3.02 (m, 8H),2.12-1.71 (series of multiplets, 17H), 1.65-1.01 (series of multiplets,13H), 0.97 (d, J=6.8 Hz, 3H), 0.94 (s, 3H), 0.73 (s, 3H); ¹³C NMR (about10% CDCl₃ in CD₃OD, 75 MHz) δ 81.89, 80.58, 77.50, 70.04, 66.71, 66.56,66.02, 47.11, 46.76, 44.20, 42.66, 40.50, 39.60, 39.40, 36.24, 36.11,35.89, 35.67, 32.28, 29.38, 29.23, 29.10, 28.94, 28.49, 26.06, 24.21,23.46, 23.30, 18.50, 12.86; HRFAB-MS (thioglycerol+Na⁺ matrix) m/e:([M+Na]⁺) 668.4271 (100%), calcd. 668.4258.

Compound CSA-13: The mesylate derived from 23 (0.19 g, 0.264 mmol) wasstirred with excess octyl amine (2 mL) at 80° C. for 12 hr. Afterremoval of octylamine in vacuo, the residue was chromatographed (silicagel, EtOAc/hexanes 1:4 with 2% Et₃ N) to afford the desired product(0.19 g, 95% yield) as a pale yellow oil. ¹H NMR (CDCl₃, 300 MHz) δ3.69-3.37 (series of multiplets, 11H), 3.26-3.00 (m, 4H), 2.61-2.53 (m,4H), 2.20-2.02 (m, 3H), 1.98-0.99 (series of multiplets, 40H), 0.92-0.85(m, 9H), 0.65 (s, 3H); ¹³C NMR (CDCl₃, 75 MHz) δ 80.60, 79.74, 76.05,64.97, 64.40, 64.28, 50.79, 50.25, 49.00, 48.90, 48.71, 46.47, 46.34,42.65, 41.96, 39.80, 35.77, 35.41, 35.27, 35.05, 33.73, 31.96, 30.25,29.76, 29.74, 29.67, 29.39, 29.05, 27.84, 27.61, 27.55, 26.70, 23.50,23.00, 22.82, 22.79, 18.06, 14.23, 12.54; HRFAB-MS (thioglycerol+Na⁺matrix) m/e: ([M+H]⁺) 755.6012 (100%), calcd. 755.6024. The triazide(0.18 g, 0.239 mmol) was dissolved in THF (10 mL) and EtOH (10 mL).Lindlar catalyst (44 mg) was added, and the suspension was shaken underH₂ (50 psi) for 12 hr. After removal of the solvent in vacuo, theresidue was purified by silica gel chromatography (CH₂Cl₂/MeOH/28%NH₃.H₂O 10:5:1, then 10:5:1.5). To the product, 1 M HCl (2 mL) and theresulting clear solution was extracted with Et₂O (2×10 mL). 20% NaOHsolution was added until the solution became strongly basic. CH₂Cl₂ (20mL, 2×10 mL) was used to extract the basic solution. The combinedextracts were dried over anhydrous Na₂SO₄, and removal of solvent invacuo gave the desired product (0.114 g, 68% yield) as a clear oil. ¹HNMR (about 20% CDCl₃ in CD₃OD, 500 MHz) δ 4.79 (bs, 7H), 3.74-3.70 (m,1H), 3.66-3.61 (m, 1H), 3.56-3.51 (m, 3H), 3.31-3.29 (m, 2H), 3.16-3.09(m, 2H), 2.88-2.72 (m, 6H), 2.59-2.51 (m, 4H), 2.18-2.07 (m, 3H),1.97-1.66 (series of multiplets, 14H), 1.62-0.97 (series of multiplets,25H), 0.95 (d, J=6.3 Hz, 3H), 0.93 (s, 3H), 0.89 (t, J=6.8 Hz, 3H), 0.70(s, 3H); ¹³C NMR (about 20% CDCl₃ in CD₃OD, 75 MHz) δ 81.82, 80.63,77.23, 67.85, 67.19, 51.20, 50.69, 47.82, 47.24, 43.92, 43.01, 41.30,40.80, 40.68, 40.22, 36.74, 36.38, 36.20, 35.87, 34.66, 34.15, 33.87,32.90, 30.54, 30.39, 30.30, 29.64, 29.03, 28.59, 28.41, 26.96, 24.37,23.65, 23.48, 18.75, 14.63, 13.09; HRFAB-MS (thioglycerol+Na⁺ matrix)m/e: ([M+H]⁺) 677.6309 (46.6%), calcd. 677.6309.

Compound CSA-46: Compound CSA-46 was prepared using the methods ofCSA-13, substituting 7-deoxycholic steroid backbone precursor in placeof cholic acid.

Compound 134: Compound CSA-13 (0.08 g, 0.12 mmol) was dissolved in CHCl₃(5 mL) and MeOH (5 mL), aminoiminosulfonic acid (0.045 g, 0.36 mmol) wasadded, and the suspension was stirred for 12 hr. The solvent was removedin vacuo, and the residue was dissolved in 1 M HCl (6 mL) and H₂O (10mL). The solution was washed with Et₂O (3×5 mL), and 20% NaOH solutionwas then added dropwise until the solution became strongly basic. Thebasic mixture was extracted with CH₂Cl₂ (4×5 mL). The combined extractswere dried over anhydrous Na₂SO₄ and concentrated in vacuo to give thedesired product (0.087 g, 91% yield) as a white glass. ¹H NMR (about 20%CDCl₃ in CD₃OD, 500 MHz) δ 4.96 (bs, 13H), 3.74-3.68 (m, 1H), 3.65-3.50(m, 4H), 3.38-3.18 (series of multiplets, 10H), 2.60-2.50 (m, 4H),2.15-1.99 (m, 3H), 1.88-1.72 (m, 14H), 1.60-0.99 (series of multiplets,25H), 0.94 (bs, 6H), 0.89 (t, J=6.6 Hz, 3H), 0.71 (s, 3H); ¹³C NMR(about 20% CDCl₃ in CD₃OD, 75 MHz) δ 159.00, 158.87, 158.72, 81.68,79.93, 76.95, 66.59, 65.93, 65.45, 50.82, 50.40, 47.64, 46.94, 43.67,42.27, 40.18, 39.25, 36.19, 35.66, 35.40, 34.21, 32.45, 30.51, 30.26,30.18, 30.10, 29.86, 29.35, 28.71, 28.15, 28.00, 26.87, 23.94, 23.44,23.23, 23.12, 18.61, 14.42, 12.98; HRFAB-MS (thioglycerol+Na⁺ matrix)m/e: ([M+H]⁺) 803.6958 (18.4%), calcd. 803.6953.

Compound CSA-15: The mesylate derived from 23 (0.092 g, 0.128 mmol) wasdissolved in DMSO (2 mL) followed by the addition of NaN₃ (0.0167 g,0.256 mmol). The suspension was heated to 70° C. for 12 hr. H₂O (20 mL)was added to the cooled suspension, and the mixture was extracted withEtOAc/hexanes (1:1) (20 mL, 3×10 mL). The combined extracts were washedwith brine (30 mL), dried over anhydrous Na₂SO₄, and concentrated invacuo to give the product (0.081 g, 95% yield) as a pale yellow oil. ¹HNMR (CDCl₃, 300 MHz) δ 3.69-3.36 (m, 11H), 3.25-3.02 (m, 6H), 2.20-2.02(m, 3H), 1.97-1.60 (m, 15H), 1.55-0.98 (m, 13H), 0.92 (d, J=6.3 Hz; 3H),0.89 (s, 3H), 0.66 (s, 3H); ¹³C NMR (CDCl₃, 75 MHz) δ 80.59, 79.77,76.03, 65.01, 64.46, 64.30, 52.12, 48.99, 48.95, 48.76, 46.44, 46.42,42.70, 41.99, 39.82, 35.56, 35.44, 35.31, 35.09, 33.09, 29.79, 29.77,29.71, 29.08, 27.88, 27.78, 27.66, 25.65, 23.53, 23.03, 22.85, 18.00,12.58; HRFAB-MS (thioglycerol+Na⁺ matrix) m/e: ([M+Na]⁺) 691.4512(100%), calcd. 691.4496. The tetraazide (0.081 g, 0.12 mmol) wasdissolved in THF (5 mL) and EtOH (10 mL). Lindlar catalyst (30 mg) wasadded, and the suspension was shaken under H₂ (50 psi) for 12 hr. Afterremoval of the solvent in vacuo, the residue was purified by silica gelchromatography (CH₂Cl₂/MeOH/28% NH₃.H₂O 5:3:1, then 2:2:1). To theproduct, 1M HCl (2 mL) was added, and the resulting solution was washedwith Et₂O (2×10 mL). 20% NaOH solution was added to the aqueous phaseuntil the solution became strongly basic. CH₂Cl₂ (10 mL, 2×5 mL) wasused to extract the basic solution. The combined extracts were driedover anhydrous Na₂SO₄, and concentration in vacuo gave the desiredproduct (0.044 g, 64% yield) as a colorless oil. ¹H NMR (about 20% CDCl₃in CD₃OD, 500 MHz) δ 4.79 (bs, 8H), 3.74-3.70 (m, 1H), 3.66-3.62 (m,1H), 3.56-3.52 (m, 3H), 3.31-3.27 (m, 2H), 3.16-3.10 (m, 2H), 2.82-2.70(m, 6H), 2.64-2.54 (m, 2H), 2.19-2.07 (m, 3H), 1.99-1.66 (series ofmultiplets, 14H), 1.58-0.96 (series of multiplets, 13H), 0.96 (d, J=6.6Hz, 3H), 0.93 (s, 3H), 0.70 (s, 3H); ¹³C NMR (about 20% CDCl₃ in CD₃OD,75 MHz) δ 81.96, 90.76, 77.33, 67.92, 67.26, 47.84, 47.33, 44.04, 43.24,43.15, 41.40, 40.91, 40.78, 40.29, 36.82, 36.48, 36.28, 35.96, 34.39,34.11, 30.59, 29.69, 29.13, 28.68, 28.64, 24.43, 23.69, 23.48, 18.77,13.06; HRFAB-MS (thioglycerol+Na⁺ matrix) m/e: ([M+H]⁺) 565.5041 (100%),calcd. 565.5057.

Example 12

This example includes a description of one or more exemplary synthesticprocedures for obtaining Compounds 203a-b, 207a-c, 209a-c, 210a-b andCSA-31.

Compounds 203a-b, 207a-c, 208a-c, 209a-c, and 210a-b: BOC-glycine wasreacted with DCC, DMAP and cholic acid derivative 201 (Scheme 11) togive triester 202a in good yield. A similar reaction incorporatingBOC-β-alanine was also successful, giving 202b. Deprotection of 202a and202b with HCl in dioxane, followed by purification (SiO₂ chromatographywith a CH₂Cl₂MeOH/NH₄OH eluent), gave triesters 203a and 203b in goodyield.

Triamides of glycine and -alanine (207a and 207b, respectively) wereformed using the same reaction conditions (Scheme 12). Triamides withα-branched amino acids could also be formed. For example, under theconditions described, a triamide with bis-BOC-lysine side chains wasformed (compound 207c). The C24 esters of 207a-c were hydrolyzed withLiOH in THF and methanol to give alcohols 208a-c. Deprotection using HClin dioxane (208a-c) gave triamides 209a-c in good yield. In addition,alcohols 208a and 208b were mesylated and reacted with benzylmethylamine. Deprotection of the resulting compounds with HCl in dioxane gavetriamides 210a and 210b (Scheme 12). Compound CSA-31 was prepared byanalogy to compounds 210a and 210b.

Example 13

This example includes a description of one or more exemplary synthesticprocedures for obtaining Compounds 302, 312-321, 324-326, 328-331 and341-343.

Compound 302: Compound 308 (5β-cholanic acid 3,7,12-trione methyl ester)was prepared from methyl cholate and pyridinium dichromate in nearquantitative yield from methyl cholate. Compound 308 can also beprepared as described in Pearson et al., J. Chem. Soc. Perkins Trans. 11985, 267; Mitra et al., J. Org. Chem. 1968, 33, 175; and Takeda et al.,J. Biochem. (Tokyo) 1959, 46, 1313. Compound 308 was treated withhydroxyl amine hydrochloride and sodium acetate in refluxing ethanol for12 hr (as described in Hsieh et al., Bioorg. Med. Chem. 1995, 3, 823),giving 309 in 97% yield.

A 250 ml three neck flask was charged with glyme (100 ml); to this wasadded 309 (1.00 g, 2.16 mmol) and sodium borohydride (2.11 g, 55.7mmol). TiCl₄ (4.0 mL, 36.4 mmol) was added to the mixture slowly undernitrogen at 0° C. The resulting green mixture was stirred at roomtemperature for 24 hours and then refluxed for another 12 h. The flaskwas cooled in an ice bath, and ammonium hydroxide (100 mL) was added.The resulting mixture was stirred for 6 hours at room temperature. Cone.HCl (60 mL) was added slowly, and the acidic mixture was stirred for 8hours. The resulting suspension was made alkaline by adding solid KOH.The suspension was filtered and the solids were washed with MeOH. Thecombined filtrate and washings were combined and concentrated in vacuo.The resulting solid was suspended in 6% aqueous KOH (100 mL) andextracted with CH₂Cl₂ (4×75 mL). The combined extracts were dried overNa₂SO₄ and solvent was removed in vacuo to give 1.14 g of a white solid.The mixture was chromatographed on silica gel (CH₂Cl₂/MeOH/NH₄OH 12:6:1)giving 302 (0.282 g, 33% yield), 3 (0.066 g, 8% yield), 4 (0.118 g, 14%yield).

Compound 302: m.p. 200-202° C.; ¹H NMR (about 10% CDCl₃ in CD₃OD, 300MHz) δ 4.81 (bs, 7H), 3.57-3.49 (m, 2H), 3.14 (t, J=3.2 Hz, 1H), 2.97(bs, 1H), 2.55-2.50 (m, 1H), 2.15-2.10 (m, 1H), 1.95-1.83 (m, 3H),1.74-0.99 (series of multiplets, 20H), 1.01 (d, J=6.4 Hz, 3H), 0.95 (s,3H), 0.79 (s, 3H); ¹³C NMR (10% CDCl₃ in CD₃OD, 75 MHz) δ 63.28, 55.01,52.39, 49.20, 48.69, 47.00, 43.24, 42.77, 41.03, 40.27, 36.82, 36.35,35.75, 35.12, 32.77, 31.36, 30.10, 28.54, 27.88, 26.96, 24.35, 23.38,18.18, 14.23, HRFAB-MS (thioglycerol+Na⁺ matrix) m/e; ([M+H]⁺) 392.3627(100%); calcd. 392.3641.

Octanyl cholate (328): Cholic acid (3.14 g, 7.43 mmol) and10-camphorsulfonic acid (0.52 g, 2.23 mmol) were dissolved in octanol(3.5 mL, 23.44 mmol). The solution was warmed to 40-50° C. in oil bathunder vacuum (about 13 mm/Hg). After 14 h, the remaining octanol wasevaporated under high vacuum. The crude product was purified viachromatography (silica gel, 5% MeOH in CH₂Cl₂) to afford the desiredproduct (2.81 g, 73% yield) as a white powder. ¹H NMR (CDCl₃, 500 MHz) δ4.06 (t, J=6.7 Hz, 2H), 3.98 (s, 1H), 3.86 (s, 1H), 3.48-3.44 (m, 1H),2.41-2.34 (m, 1H), 2.28-2.18 (m, 3H), 1.98-1.28 (series of multiplets,35H), 0.99 (d, J=3.3 Hz, 3H), 0.90 (s, 3H), 0.89 (t, J=7 Hz, 3H), 0.69(s, 3H); ¹³C NMR (CDCl₃, 75 MHz) δ 154.38, 73.18, 72.14, 68.63, 56.07,50.02, 49.32, 47.07, 46.74, 41.96, 41.67, 39.84, 39.76, 35.66, 35.45,34.95, 34.86, 34.15, 32.97, 32.91, 31.65, 31.11, 30.68, 28.39, 27.78,26.66, 26.52, 25.82, 25.70, 25.54, 25.15, 24.95, 23.45, 22.69, 17.77,12.71; HRFAB-MS (thioglycerol+Na⁺ matrix) m/e: ([M+Na]⁺) 543.4015(100%), calcd. 543.4026.

Representative synthesis of compounds 329-331: Octanyl cholate (328)(0.266 g, 0.511 mmol), N-t-Boc-glycine (0.403 g, 2.298 mmol), DCC (0.474g, 2.298 mmol) and DMAP (0.0624 g, 0.051 mmol) were mixed in CH₂Cl₂ (15mL) for 3 h. The resulting white precipitate was removed by filtration.The filtrate was concentrated, and the product was purified bychromatography (silica gel, EtOAc/Hexane 1:2) to afford the desiredproduct (0.481 g, 95% yield) as a white powder. Compound 329 ¹H NMR(CDCl₃, 300 MHz) δ 5.18 (br, 3H), 5.01 (s, 1H), 4.61 (m, 1H), 4.04 (t,J=6.5 Hz, 2H), 3.97-3.88 (series of multiplets, 6H), 2.39-2.15 (seriesof multiplets, 2H), 2.06-1.02 (series of multiplets, 35H), 1.46 (s,18H), 1.45 (s, 9H), 0.93 (s, 3H), 0.88 (t, J=6.7 Hz, 3H), 0.81 (d, J=6Hz, 3H), 0.74 (s, 3H); ¹³C NMR (CDCl₃, 75 MHz) δ174.26, 170.19, 169.9,169.78, 155.87, 155.67, 79.95, 76.47, 75.167, 72.11, 64.55, 47.40,45.28, 43.17, 42.86, 40.82, 37.94, 34.71, 34.63, 34.43, 31.86, 31.340,31.20, 30.76, 29.29, 29.25, 28.80, 28.72, 28.42, 28.06, 27.96, 27.19,26.81, 26.29, 26.012, 25.66, 22.87, 22.71, 22.57, 17.55, 14.18, 12.27;HRFAB-MS (thioglycerol+Na⁺ matrix) m/e: ([M+Na]⁺) 1014.6261 (100%),calcd. 1014.6242. Compound 330: ¹H NMR (CDCl₃, 500 MHz) δ 5.10 (s, 1H),4.92 (d, J=2.44 Hz, 1H), 4.55 (m, 1H), 4.00 (t, J=6.8 Hz, 2H), 3.39-3.33(series of multiplets, 6H), 2.595-2.467 (series of multiplets, 6H),2.31-2.12 (series of multiplets, 2H), 2.01-1.00 (series of multiplets,37H), 1.39 (s, 27H), 0.88 (s, 3H), 0.84 (t, J=6.8 Hz, 3H), 0.76 (d,J=6.3 Hz, 3H), 0.69 (s, 3H); ¹³C NMR (CDCl₃, 75 MHz) δ 174.16, 172.10,171.78, 171.67, 155.95, 79.45, 75.67, 74.21, 71.10, 64.63, 47.79, 45.27,43.52, 40.97, 37.92, 36.35, 35.14, 35.05, 34.90, 34.71, 34.46, 31.91,31.45, 30.95, 29.35, 29.31, 28.96, 28.78, 28.56, 28.55, 27.22, 26.98,26.269, 25.71, 23.00, 22.77, 22.64, 17.75, 14.24, 12.39; HRFAB-MS(thioglycerol+Na⁺ matrix) m/e: ([M+Na]⁺) 1056.6702 (100%), calcd.1056.6712. Compound 331 ¹³C NMR (CDCl₃, 125 MHz) δ174.00, 172.75,172.41, 172.30, 156.03, 79.00, 75.28, 73.79, 70.77, 64.39, 47.43, 45.04,43.21, 40.76, 40.00, 39.93, 37.78, 34.74, 34.62, 34.23, 32.19, 32.01,31.70, 31.24, 30.77, 29.13, 29.10, 28.67, 38.58, 28.38, 25.86, 25.37,22.56, 22.38, 17.51, 14.05, 12.13; HRFAB-MS (thioglycerol+Na⁺ matrix)m/e: ([M+Na]⁺) 1098.7181 (100%), calcd. 1098.7181.

Representative synthesis of compounds 341-343: To compound 329 (0.463 g,0.467 mmol) was added HCl in dioxane (0.3 mL, 4.0 M). After stirring themixture for 30 min, the excess HCl and solvent were removed in vacuo.The product was isolated, after chromatography (silica gel,CH₂Cl₂/MeOH/NH₃.H₂O 10:1.2:0.1) as a (0.271 g, 84%) pale oil. Thetrihydrochloride salt of 341 was prepared by addition of HCl in dioxaneand evaporation of excess HCl and dioxane in vacuo giving a whitepowder. Compound 341: ¹H NMR (CDCl₃ with about 10% CD₃OD, 500 MHz) δ5.16 (s, 1H), 4.99 (t, J=3.6 Hz, 1H), 4.61 (m, 1H), 4.04 (t, J=6.8 Hz,2H), 3.51-3.36 (m, 6H), 2.34-2.15 (m, 2H), 2.00-1.05 (series ofmultiplets, 40H), 0.93 (s, 3H), 0.88 (t, J=7.1 Hz, 3H), 0.80 (d, J=3.2Hz, 3H), 0.74 (s, 3H); ¹³C NMR (CDCl₃ and about 10% CD₃OD, 75 MHz) δ174.32, 173.92, 173.81, 76.08, 74.67, 71.61, 64.73, 47.64, 45.39, 44.41,43.49, 40.97, 37.99, 34.99, 34.77, 34.71, 34.52, 31.96, 31.54, 31.35,30.96, 29.39, 29.36, 29.02, 28.82, 27.32, 27.11, 26.11, 25.83, 23.01,22.82, 22.69, 17.79, 14.28, 12.41; HRFAB-MS (thioglycerol+Na matrix)m/e: ([M+Na]⁺) 714.4651 (100%), calcd. 714.4669. Compound 342: ¹H NMR(CDCl₃ and about 10% CD₃OD, 300 MHz) δ 5.142 (s, 1H), 4.96 (d, J=2.7 Hz,1H), 4.60, (m, 1H), 4.04 (t, J=6.6 Hz, 2H), 3.07-2.95 (series ofmultiplets, 6H), 2.56-2.43 (series of multiplets, 6H), 2.38-2.13 (seriesof multiplets, 2H), 2.07-1.02 (series of multiplets, 36H), 0.92 (s, 3H),0.88 (t, J=6.6 Hz, 3H), 0.82 (d, J=6.6 Hz, 3H), 0.73 (s, 3H); ¹³C NMR(CDCl₃ and CD₃OD, 75 MHz) δ 174.29, 172.29, 171.98, 171.92, 75.52,74.09, 70.98, 64.67, 47.78, 45.26, 43.52, 40.98, 38.73, 38.62, 38.35,38.07, 38.03, 37.99, 35.01, 34.81, 34.77, 34.49, 31.92, 31.50, 31.40,30.99, 29.36, 29.33, 28.93, 28.80, 27.43, 26.96, 26.08, 25.56, 23.07,22.79, 22.62, 17.73, 14.25, 12.34; HRFAB-MS (thioglycerol+Na⁺ matrix)m/e: ([M+Na]⁺) 714.4651 (100%), calcd. 714.4669. Compound 343: ¹H NMR(CDCl₃ and CD₃OD, 500 MHz) δ 5.12 (s, 1H) 4.93 (s, 1H), 4.59 (m, 1H),4.04 (t, J=7 Hz, 2H), 2.79-2.69 (series of multiplets, 6H),2.4621-2.2999 (series of multiplets, 6H), 2.2033-1.0854 (series ofmultiplets, 42H), 0.94 (s, 2H), 0.91 (s, 1H), 0.88 (t, J=7 Hz, 3H), 0.82(d, J=6.4 Hz, 3H), 0.75 (s, 3H); ¹³C NMR (CDCl₃ and CD₃OD, 75 MHz) δ174.70, 171.97, 171.86, 171.75, 76.10, 74.55, 71.56, 64.85, 47.96,45.31, 43.37, 40.87, 38.09, 34.86, 34.80, 34.73, 34.46, 32.84, 32.62,32.27, 31.87, 31.75, 31.42, 31.08, 29.31, 29.28, 29.26, 28.78, 28.73,27.38, 26.91, 26.05, 25.37, 23.24, 23.15, 22.95, 22.74, 22.71, 22.43,17.78, 14.11, 12.28; HRFAB-MS (thioglycerol+Na⁺ matrix) m/e: ([M+Na]⁺)798.5624 (100%), calcd. 798.5609.

Benzyl cholate (312): Cholic acid (4.33 g, 10.62 mmol) and10-caphorsulfonic acid (0.493 g, 2.21 mmol) were dissolved in benzylalcohol (1.97 mL, 19.3 mmol). The suspension was heated to 50° C. in oilbath and stirred under vacuum (about 13 mm/Hg) for 16 h. Excess benzylalcohol was removed in vacuo, and the crude product was chromatographed(silica gel, 5% MeOH in CH₂Cl₂) to give the desire product as a whitepowder (4.23 g, 81% yield). ¹H NMR (CDCl₃, 500 MHz) δ 7.34-7.33 (m, 5H),5.10 (d, J=1.5 Hz, 2H), 3.92 (s, 1H), 3.81 (s, 1H), 3.42 (s, 1H), 3.40(br, m, 3H), 2.44-2.38 (m, 1H), 2.31-2.25 (m, 1H), 2.219 (t, J=12 Hz,2H), 0.96 (d, J=5.5 Hz, 3H), 0.86 (s, 3H), 0.63 (s, 3H); ¹³C NMR (CDCl₃,125 MHz) δ174.25, 136.30, 128.66, 128.63, 128.32, 128.28, 128.24, 73.18,71.98, 68.54, 66.18, 47.14, 46.56, 41.69, 39.65, 35.51, 35.37, 34.91,34.84, 31.49, 31.08, 30.50, 28.31, 27.62, 26.47, 23.35, 22.65, 22.60,17.42, 12.63, 12.57; HRFAB-MS (thioglycerol+Na⁺ matrix) m/e: ([M+Na]⁺)521.3235 (100%), calcd. 521.3242.

Representative synthesis of compounds 313-315: Benzyl cholate (312)(0.248 g, 0.499 mmol), N-t-Boc-glycine (0.404 g, 2.30 mmol), DCC (0.338g, 1.49 mmol) and DMAP (0.051 g, 0.399 mmol) were added to CH₂C₂ (15mL), and the suspension was stirred for 16 h. The resulting whiteprecipitate was removed by filtration, and the filtrate wasconcentrated. The product was obtained after chromatorgraphy (silicagel, EtOAc/Hexane 0.6:1) as a white powder (0.329 g, 68%). Compound 313:¹H NMR (CDCl₃, 300 MHz) δ 7.34-7.33 (m, 5H), 5.16 (s, 1H), 5.08 (dd,J=22.5 Hz, 12.3 Hz, 4H), 5.00 (s, 1H), 4.60 (m, 1H), 4.04-3.81 (seriesof multiplets, 6H), 2.43-1.01 (series of multiplets, 25H), 1.46 (s, 9H),1.44 (s, 18H), 0.92 (s, 3H), 0.797 (d, J=5.7 Hz, 3H), 0.69 (s, 1H); ¹³CNMR (CDCl₃, 75 MHz) δ 173.99, 170.25, 170.05, 169.85, 155.73, 136.19,128.69, 128.45, 128.35, 80.06, 77.65, 77.23, 76.80, 76.53, 75.24, 72.19,66.29, 47.46, 45.35, 43.24, 42.91, 40.89, 38.00, 34.79, 34.66, 34.49,31.43, 31.25, 30.77, 28.88, 28.40, 27.23, 26.89, 25.74, 22.94, 22.65,17.61, 12.32; FAB-MS (thioglycerol+Na⁺ matrix) m/e: ([M+Na]⁺) 992.5468(100%), calcd. 992.5460.

Representative synthesis of compounds 316-318: Compound 313 (0.505 g,0.520 mmol) and Pd (5 wt. % on active carbon, 0.111 g, 0.0521 mmol) wereadded to MeOH (5 mL). The suspension was stirred under H₂ (50 psi) for20 hours. The solids were removed by filtration and the filtrate wasconcentrated. Purification of the product via chromatography (silicagel, 5% MeOH in CH₂Cl₂) gave a white powder (0.450 g, 98% yield).Compound 316: ¹H NMR (CDCl₃, 500 MHz) δ 5.20 (s, 1H), 5.12 (br., 2H),4.92 (s, 1H), 4.55 (m, 1H), 3.98-3.83 (series of multiplets, 6H),2.30-2.13 (series of multiplets, 2H), 1.96-0.98 (series of multiplets,30H), 1.40 (s, 9H), 1.39 (s, 18H), 0.87 (s, 3H), 0.76 (d, J=6.3 Hz, 3H),0.68 (s, 3H); ¹³C NMR (CDCl₃ 75 MHz) δ174.11, 165.60, 165.41, 165.22,151.28, 151.14, 75.48, 75.26, 71.81, 70.57, 67.50, 45.95, 42.58, 40.65,38.52, 38.16, 36.17, 33.28, 30.01, 29.78, 26.71, 26.42, 25.95, 24.16,23.78, 23.40, 23.31, 22.55, 22.16, 21.03, 18.23, 17.93, 12.91, 7.61;FAB-MS (thioglycerol+Na⁺ matrix) m/e: ([M+Na]⁺) 902.4997 (21%), calcd.902.4990.

Representative synthesis of compounds 319-321: Compound 316 (0.375 g,0.427 mmol), DCC (0.105 g, 0.512 mmol) and DMAP (0.062 g, 0.512 mmol)and N,N-dimethylethanolamine (0.09 ml, 0.896 mmol) were added to CH₂Cl₂(15 mL). The mixture for 16 h, and solvent and excessN,N-dimethylethanolamine were removed in vacuo. The product was purifiedvia chromatography (silica gel EtOAc/hexane/Et3 N, 12:10:0.6) giving awhite powder (0.330 g, 82% yield). ¹H NMR (CDCl₃ and about 10% CD₃OD,500 MHz) δ 5.18 (s, 1H), 5.00 (s, 1H), 4.19 (t, J=5.0 Hz, 2H), 3.92 (s,3H), 3.81 (s, 3H), 2.62 (t, J=10 Hz, 2H), 2.30 (s, 6H), 1.47 (s, 9H),1.47 (s, 1H), 1.45 (s, 1H), 2.12-1.05 (series of multiplets, 27H), 0.96(s, 3H), 0.84 (d, J=10.5 Hz, 3H), 0.78 (s, 3H); ¹³C NMR (CDCl₃ and about10% CD₃OD, 125 MHz) 5174.19, 170.05, 169.87, 156.21, 79.36, 79.27,76.06, 76.90, 71.80, 61.19, 57.04, 46.88, 44.87, 44.67, 44.53, 42.78,42.15, 42.01, 40.43, 37.47, 34.32, 34.11, 33.92, 33.35, 33.25, 30.74,30.56, 30.16, 28.40, 27.67, 27.62, 26.73, 26.19, 25.18, 25.10, 24.72,24.49, 22.29, 21.81, 16.76, 11.56; FAB-MS (thioglycerol+Na⁺ matrix) m/e:([M+Na]⁺) 973.5723 (100%), calcd. 973.5725. The white solid from theprevious reaction (0.680 g, 0.714 mmol) and MeI (1 M in CH₂Cl₂, 1.5 mL)were stirred together for 2 h: The solvent and excess MeI were removedin vacuo giving a white solid (0.812 g about 100%). The product wascarried on without further purification.

Representative synthesis of compounds 324-326: Compound 319 (0.812 g,0.714 mmol) was dissolved in CH₂Cl₂ (5 mL) and trifluoroacetic acid (0.5mL) was added. The mixture was stirred for 16 min. The solvent andexcess acid were removed in vacuo, and the resulting oil waschromatographed (silica gel, CH₂Cl₂/MeOH/NH₃.H₂O 4:4:1) to give thedesired product as a pale glass (0.437 g, 90% yield). Addition of HCl (2M in ethyl ether, 2.5 mL) gave the trihydrochloride salt of 324 as apale yellow powder. Compound 324: ¹H NMR (50% CDCl₃, 50% CD₃OD, 300 MHz)δ 5.43 (s, 1H), 5.24 (s, 1H), 4.84 (m, 1H), 4.66 (m, 2H), 4.16-3.96(series of multiplets, 6H), 3.88 (m, 2H), 3.37 (s, 9H), 0.67 (s, 3H),0.59 (d, J=6.3 Hz, 3H), 0.56 (s, 3H); ¹³C NMR (50% CDCl₃, 50% CD₃OD, 75MHz) δ 173.47, 167.06, 167.01, 166.70, 78.01, 76.49, 73.78, 64.98,57.67, 53.36, 47.49, 46.99, 45.61, 43.28, 40.83, 40.23, 40.10, 37.69,34.80, 34.48, 34.28, 31.03, 30.63, 30.44, 28.94, 27.05, 26.56, 25.50,22.53, 21.56, 16.95, 11.37; FAB-MS (thioglycerol+Na⁺ matrix) m/e:([M-I]⁺) 665.4475 (85.6%), cacld 665.4489. Compounds 325 and 326 provedtoo unstable to chromatograph using the basic eluent used for thepurification of 324. Consequently, 325 and 326 were prepared bydeprotection of 320 and 321 using HCl (2 M in diethyl ether), followedby tituration with ethyl acetate. The compounds were then used withoutfurther purification. ¹H NMR spectroscopy indicated that compounds 325and 326 were >95% pure. Compound 325: ¹H NMR (50% CDCl₃, 50% CD₃OD, 500MHz) δ 5.21 (s, 1H), 5.02 (d, J=4 Hz, 1H), 4.64 (m, 1H), 4.53 (m, 2H),3.74 (m, 2H), 3.31-3.01 (series of multiplets, 6H), 3.23 (s, 9H),2.96-2.73 (series of multiples, 6H), 2.51-2.44 (m, 1H), 2.35-2.29 (m,1H), 2.14-1.09 (series of multiplets, 26H), 0.99 (s, 3H), 0.85 (d, J=6.5Hz, 3H), 0.80 (s, 3H); ¹³C NMR (50% CDCl₃, 50% CD₃OD, 125 MHz) δ 172.77,169.88, 169.56, 169.50, 75.94, 74.44, 71.57, 64.31, 56.94, 52.92, 46.78,44.59, 42.70, 40.21, 37.16, 34.80, 34.72, 34.66, 34.05, 34.00, 33.78,33.62, 30.95, 30.91, 30.81, 30.41, 29.96, 29.81, 28.20, 26.37, 26.06,24.74, 24.24, 22.04, 21.13, 16.54, 10.97; FAB-MS (thioglycerol+Na⁺matrix) m/e: ([M-I]⁺) 707.4958 (25.6%), cacld 707.4958. Compound 326: ¹HNMR (50% CDCl₃, 50% CD₃OD, 500 MHz) δ 5.12 (s, 1H), 4.94 (d, J=2.5 Hz,1H), 4.56 (m. 1H), 4.51 (t, J=2.3 Hz, 2H), 3.74 (m, 2H), 3.23 (s, 9H),3.05-3.01 (m, 4H), 2.98 (t, J=7.5 Hz, 2H), 2.63-2.43 (series ofmultiplets, 6H), 2.31-2.24 (series of multiplets, 2 H), 2.07-1.87(series of multiplets, 12H), 1.17-1.05 (series of multiplets, 23H), 0.94(s, 3H), 0.82 (d, J=6.0 Hz, 3H), 0.76 (s, 3H); ¹³C NMR (50% CDCl₃, 50%CD₃OD, 125 MHz) 5171.87, 169.79, 169.59, 169.50, 76.12, 74.70, 71.65,65.57, 65.08, 64.40, 57.68, 53.74, 52.78, 45.33, 43.54, 41.04, 39.12,37.92, 43.85, 34.72, 34.56, 34.34, 32.30, 31.47, 31.27, 30.87, 30.58,29.03, 27.053, 26.84, 25.51, 24.95, 24.91, 22.87, 22.82, 22.65, 21.93,17.31, 11.81; FAB-MS (thioglycerol+Na⁺ matrix) m/e: ([M-I]⁺) 749.5432(100%), cacld 749.5436.

Example 14

This example includes data indicating the stability of Compounds 352-354under acidic, neutral and basic conditions.

Compounds 352-354 were dissolved in 50 mM phosphate buffered water (pH2.0, 7.0 or 12.0) at approximately 10 mM concentrations. The structuresof compounds 352-354 are given in FIG. 9. Decomposition of the compoundswas observed via HPLC (cyano-silica column, 0.15% TFA water-acetonitrilegradient elution). Table 15 shows the stabilities (half-lives) ofcompounds 352-354 in phosphate buffer at room temperature, pH 2.0, pH7.0 and pH 12.0. These compounds were used since they contain achromophore that facilitated monitoring of decomposition by absorptionmethods common in the HPLC apparatus used.

At low pH, the amines are expected to be protonated and the compoundsshowed relative stability. At higher pH, the amines were less stronglyprotonated and became involved in ester hydrolysis. The γ-aminobutyricacid-derived compound was especially susceptible to hydrolysis,presumably yielding pyrrolidone. In general, the compounds are believedto hydrolyse to give cholic acid, choline or octanol, and glycine,beta-alanine, or pyrrolidone, depending on the particular compound.

Decomposition through ester hydrolysis yielded compounds that were lesspolar and easily separable from the starting compounds. Initially, onlyone benezene-containing decomposition product was observed; at longerreaction times, two other decomposition products were observed whichpresumably corresponded to sequential ester hydrolysis.

Example 15

This example includes a description of additional exemplary syntheticprocedures for producing compounds of formula I. In one example,hydroxyl groups on cholic acid can be converted into amine groups asdescribed in in Hsieh et al. (Synthesis and DNA Binding Properties ofC3-, C12-, and C24-Substituted Amino-Steroids Derived from Bile Acids,Biorganic and Medicinal Chemistry, 1995, vol. 6, 823-838).

Compounds of formula I prepared as shown in the following Scheme.

Description of the steroid starting materials shown above can be foundin Dictionary of Steroids, Hill, R. R.; Kirk, D. N.; Makin, H. L. J.;Murphy. G. M., eds Chapman and Hall: New York, 1991.

1-26. (canceled)
 27. A detectably labelled cationic steroidalantimicrobial (CSA) for use in detecting an infection, or diagnosing asubject having or at risk of having an infection, the detectablylabelled CSA comprising: a steroid backbone; a plurality of amine orguanidine groups covalently attached to the steroid backbone; and adetectable label incorporated into the structure of the CSA orcovalently linked or conjugated to the CSA, wherein the detectable labelcomprises a radioisotope, a metal, or a metal oxide, wherein the CSA isconfigured to bind to cell membranes of viruses, fungi, and bacteria.28. The detectably labelled CSA of claim 27, wherein the CSA is acompound having a structure of Formula V, or a pharmaceuticallyacceptable salt thereof:

where: each of fused rings A, B, C, and D is independently saturated, oris fully or partially unsaturated, provided that at least two of A, B,C, and D are saturated; each of m, n, p, and q is independently 0 or 1;each of R₁ through R₄, R₆, R₇, R₁₁, R₁₂, R₁₅, R₁₆, R₁₇, and R₁₈ isindependently selected from the group consisting of hydrogen, hydroxyl,substituted or unsubstituted alkyl, hydroxyalkyl, alkyloxyalkyl,alkylcarboxyalkyl, alkylaminoalkyl, alkylamino-alkylamino,alkylamino-alkylamino-alkylamino, substituted or unsubstitutedaminoalkyl, substituted or unsubstituted aryl, substituted orunsubstituted arylaminoalkyl, haloalkyl, alkenyl, alkynyl, oxo, linkinggroup attached to a second steroid, substituted or unsubstitutedaminoalkyloxy, substituted or unsubstituted aminoalkyloxyalkyl,substituted or unsubstituted aminoalkylcarboxy, substituted orunsubstituted aminoalkylaminocarbonyl, substituted or unsubstitutedaminoalkylcarboxamido, H₂N—HC(Q₅)-C(O)—O—, H₂N—HC(Q₅)-C(O)—N(H)—,azidoalkyloxy, cyanoalkyloxy, P.G.-HN—HC(Q₅)-C(O)—O—, guanidinoalkyloxy,quaternary ammonium alkylcarboxy, and guanidinoalkylcarboxy, where Q₅ isa side chain of an amino acid and P.G. is an amino protecting group: andeach of R₅, R₈, R₉, R₁₀, R₁₃, and R₁₄ is independently deleted when oneof fused rings A, B, C, or D is unsaturated so as to complete thevalency of the carbon atom at that site, or selected from the groupconsisting of hydrogen, hydroxyl, substituted or unsubstituted alkyl,hydroxyalkyl, alkyloxyalkyl, substituted or unsubstituted aminoalkyl,substituted or unsubstituted aryl, haloalkyl, alkenyl, alkynyl, oxo, alinking group attached to a second steroid, substituted or unsubstitutedaminoalkyloxy, substituted or unsubstituted aminoalkylcarboxy,substituted or unsubstituted aminoalkylaminocarbonyl,H₂N—HC(Q₅)-C(O)—O—, H₂N—HC(Q₅)-C(O)—N(H)—, azidoalkyloxy, cyanoalkyloxy,P.G.-HN—HC(Q₅)-C(O)—O—, guanidinoalkyloxy, and guanidinoalkylcarboxy,where Q₅ is a side chain of an amino acid and P.G. is an aminoprotecting group, provided that at least three of R₁ through R₄, R₆, R₇,R₁₁, R₁₂, R₁₅, R₁₆, R₁₇, and R₁₈ are disposed on the same face of thering system and are independently selected from the group consisting ofsubstituted or unsubstituted aminoalkyl, substituted or unsubstitutedaminoalkyloxy, alkylcarboxyalkyl, alkylamino-alkylamino,alkylamino-alkylamino-alkylamino, substituted or unsubstitutedaminoalkylcarboxy, substituted or unsubstituted arylamino-alkyl,substituted or unsubstituted aminoalkyloxy-aminoalkylaminocarbonyl,substituted or unsubstituted aminoalkylarainocarbonyl, substituted orunsubstituted aminoalkylcarboxamido, quaternary ammonium alkylcarboxy,H₂NHC(Q₅)-C(O)—O—, H₂N—HC(Q₅)-C(O)—N(H)—, azidoalkyloxy, cyanoalkyloxy,P.G.-HN—HC(Q₅)-C(O)—O—, guanidinoalkyloxy, and guanidinoalkylcarboxy.29. The detectably labelled CSA of claim 28, wherein the detectablelabel comprises a radioisotope of carbon, hydrogen, nitrogen, oxygen, orsulfur included within the structure of the CSA.
 30. The detectablylabelled CSA of claim 28, wherein the detectable label is a radioisotopecovalently linked or conjugated to the CSA and selected from the groupconsisting of C, N, O, H, S, Cu, Fe, Ga, Ti, Sr, Y, Tc, In, Pm, Gd, Sm,Ho, Lu, Re, At, Bi, and Ac.
 31. The detectably labelled CSA of claim 28,wherein the detectable label is a radioisotope covalently linked orconjugated to the CSA and selected from the group consisting of ³H, ¹⁰B,¹¹C, ¹⁴C, ¹³N, ¹⁵O, ¹⁸O, ¹⁸F, ³²P, ³⁵S, ³⁵Cl, ⁴⁵Ti, ⁴⁶Sc, ⁵¹Cr, ⁵²Fe,⁵⁹Fe, ⁵⁷Co, ⁶⁰Co, ⁶⁰Cu, ⁶¹Cu, ⁶²Cu, ⁶⁴Cu, ⁶⁷Cu, ⁶⁷Ga, ⁶⁸Ga, ⁷²As, ⁷⁶Br,⁷⁷Br, ^(81m)Kr, ⁸²Rb, ⁸⁵Sr, ⁸⁹Sr, ⁸⁶Y, ⁹⁰Y, ⁹⁵Nb, ^(94m)Tc ^(99m)Tc,⁹⁷Ru, ¹⁰³Ru, ¹⁰⁵Rh, ¹⁰⁹Cd, ¹¹¹In, ¹¹³Sn, ^(113m)In, ¹¹⁴In, ¹²³I ¹²⁴I,¹²⁵I, ¹³³Xe, ¹³⁷Cs, ¹⁴⁰La, ¹⁴¹Ce, ¹⁴⁹Pm, ¹⁵³Gd, ¹⁵⁷Gd, ¹⁵³Sm, ¹⁶¹Tb,¹⁶⁶Dy, ¹⁶⁶Ho, ¹⁶⁹Er, ¹⁶⁹Y, ¹⁷⁵Yb, ¹⁷⁷Lu, ¹⁸⁶Re, ¹⁸⁸Re, ²⁰¹Ti, ²⁰³Pb,²¹¹At, ²¹²Bi, ²²⁵Ac, and ²²⁶Ra.
 32. The detectably labelled CSA of claim31, wherein the radioisotope is selected from the group consisting of^(99m)Tc, ¹⁸⁶Re, and ¹⁸⁸Re.
 33. The detectably labelled CSA of claim 31,wherein the radioisotope is a beta emitter selected from the groupconsisting of ^(99m)Tc, ⁶⁰Co, ¹³⁷Cs, and ²²⁶Ra.
 34. The detectablylabelled CSA of claim 27, wherein the detectable labelled CSA isconfigured to bind to cell membranes of at least bacterium and at leastone virus: (a) wherein the at least bacterium is selected from the groupconsisting of Bordetella pertussis, Borrelia burgdorferi, Brucellaabortus, Brucella canis, Brucella melitensis, Brucella suis,Campylobacter jejuni, Escherichia coli, Francisella tularensis,Haemophilus influenzae, Helicobacter pylori, Legionella pneumophila;Leptospira interrogans, Neisseria gonorrhoeae, Neisseria meningitides,Pseudomonas aeruginosa, Rickettsia rickettsii, Salmonella typhi,Salmonella typhimurium, Shigella sonnei, Treponema pallidum, Vibriocholerae, Yersinia pestis, mycobacterium, listeria monocytogenes,helicobacter, bordetella, streptococcus, salmonella, Chlamydia,Clostridium botulinum, Clostridium difficile, Clostridium perfringens,Clostridium tetani, Corynebacterium diphtheriae, Enterococcus faecalis,Enterococcus faecum, Listeria, monocytogenes, Staphylococcus aureus,Staphylococcus epidermidis, Staphylococcus saprophyticus, Streptococcusagalactiae, Streptococcus pneumoniae, Streptococcus pyogenes, Chlamydiapneumoniae, Chlamydia psittaci, Chlamydia trachomatis, Mycobacteriumleprae, Mycobacterium tuberculosis, Mycoplasma pneumoniae; and (b)wherein the at least virus is selected from the group consisting ofpoxvirus, herpesvirus, hepatitis virus, immunodeficiency virus,flavivirus, papilloma virus (PV), polyoma virus, rhabdovirus, myxovirus,arenavirus, coronavirus, adenovirus, reovirus, picornavirus, togavirus,bunyavirus, parvovirus, and retrovirus.
 35. The detectably labelled CSAof claim 27, wherein the detectably labelled CSA is configured to bedetected by magnetic resonance spectroscopy (MRS), magnetic resonanceimaging (MRI), positron-emission tomography (PET), gamma-scintigraphy,computed tomography (CT), Computed Axial Tomography (CAT), or singlephoton emission tomography (SPECT).
 36. A method of detecting aninfection, or diagnosing a subject having or at risk of having aninfection, comprising: administering the detectably labelled CSA ofclaim 27 to the subject under conditions whereby the detectably labelledCSA can bind to cell membranes of viruses, fungi, and bacteria that arecausative of infection and present in the subject, and detecting thedetectably labelled CSA in the subject, including its location, toascertain the presence or absence of an infection caused by any one ofviruses, fungi, and bacteria, thereby detecting the infection, ordiagnosing the subject as having or not having an infection.
 37. Themethod of claim 36, wherein detecting the radiolabeled CSA in thesubject comprises imaging the radiolabeled CSA.
 38. The method of claim36, wherein the CSA is selected from CSA-1 through CSA-400.
 39. Themethod of claim 36, wherein the CSA is selected from CSA-13, CSA-107,and CSA-100.
 40. The method of claim 36, wherein the method detects thepresence or absence, type, kind, location, extent, severity, orprogression of the infection.
 41. The method of claim 36, wherein theinfection is a bacterial infection selected from the group consisting ofBordetella, Bordetella pertussis; Borrelia, Borrelia burgdorferi;Brucella, Brucella abortus, Brucella canis, Brucella melitensis,Brucella suis, Campylobacter, Campylobacter jejuni; Escherichia,Escherichia coli; Francisella, Francisella tularensis; Haemophilus,aemophilus luenzae; icobacter, Helicobacter pylori; Legionella,Legionella pneumophila; Leptospira, Leptospira interrogans; Neisseria,Neisseria gonorrhoeae, Neisseria meningitides; Pseudomonas, Pseudomonasaeruginosa; Rickettsia Rickettsia rickettsii, Salmonella, Salmonellatyphi, Salmonella typhimurium; Shigella Shigella sonnei; Treponema,Treponema pallidum; Vibrio, Vibrio cholerae; Yersinia, Yersinia pestisamycobacterium, listeria monocytogenes, helicobacter, bordetella,streptococcus, salmonella, Chlamydia, Clostridium, Clostridiumbotulinum, Clostridium difficile, Clostridium perfringens, Clostridiumtetani; Corynebacterium, Corynebacterium diphtheriae; Enterococcus,Enterococcus faecalis, Enterococcus faecum; Listeria Listeria,monocytogenes; Staphylococcus, Staphylococcus aureus; Staphylococcusepidermidis, Staphylococcus saprophyticus; Streptococcus, Streptococcusagalactiae; Streptococcus pneumoniae; Streptococcus pyogenes, Chlamydia,Chlamydia pneumoniae, Chlamydia psittaci, Chlamydia trachomatis;Mycobacterium, Mycobacterium leprae, Mycobacterium tuberculosis;Mycoplasma, Mycoplasma pneumoniae.
 42. The method of claim 36, whereinthe infection is a viral infection selected from the group consisting ofa poxvirus, herpesvirus, hepatitis virus, immunodeficiency virus,flavivirus, papilloma virus (PV), polyoma virus, rhabdovirus, amyxovirus, an arenavirus, a coronavirus, adenovirus, reovirus,picornavirus, togavirus, bunyavirus, parvovirus, and retrovirus.
 43. Themethod of claim 36, wherein the infection is an infection of skin,dermis, breast, lung, nasopharynx, nose or sinuses, thyroid, head, neck,brain, spine, adrenal gland, thyroid, lymph, blood, gastrointestinaltract, genito-urinary tract, kidney, pancreas, adrenal gland, liver,bone, bone marrow, heart, muscle, or hematopoetic system.
 44. The methodof claim 36, wherein detecting comprises magnetic resonance spectroscopy(MRS), magnetic resonance imaging (MRI), positron-emission tomography(PET), gamma-scintigraphy, computed tomography (CT), Computed AxialTomography (CAT), or single photon emission tomography (SPECT).
 45. Anex vivo or in vitro method of detecting an infection, or diagnosing asubject having or at risk of having an infection, comprising: contactingthe detectably labeled CSA of claim 27 to a biological sample from asubject under conditions whereby the detectably labeled CSA can bind tocell membranes of viruses, fungi, and bacteria that are causative ofinfection and present in the biological sample; and detecting thelabeled CSA in the sample to ascertain the presence or absence of aninfection in the sample, thereby of detecting an infection, ordiagnosing the subject as having or not having an infection.
 46. Themethod of claim 45, wherein the biological sample comprises at least oneof mucus, saliva, feces, blood, serum, plasma, cerebrospinal fluid,urine, or placenta blood, skin, dermis, breast, lung, nasopharynx, noseor sinuses, thyroid, head, neck, brain, spine, adrenal gland, thyroid,lymph, gastrointestinal tract, genito-urinary tract, kidney, pancreas,adrenal gland, liver, bone, bone marrow, heart, muscle, or a sample ofthe hematopoetic system.